Processes for the manufacture of macrocyclic depsipeptides and new intermediates

ABSTRACT

The invention relates to a method or process for the chemical manufacture of depsipeptides of the formula I, 
                         
wherein the symbols have the meaning defined in the description, to new intermediates and their manufacture, as well as related invention embodiments.

This application is a divisional application of U.S. patent applicationSer. No. 13/449,937, filed on Apr. 18, 2012, which claims benefit under35 U.S.C. §119(e) of U.S. Provisional Application No. 61/477,319 filedApr. 20, 2011; the contents of which is incorporated herein by referencein their entirety.

SUMMARY OF THE INVENTION

The invention relates to a method or process for the manufacture ofmacrocyclic depsipeptides, to new intermediates and their manufacture,as well as related invention embodiments.

BACKGROUND OF THE INVENTION

Cyclic depsipeptides have numerous uses in pharmacology. As an example,the depsipeptides disclosed in WO2009/024527 are useful for treatment ofvarious diseases. For example, the compound of formula II mentioned inWO2009/024527 is useful for the treatment and prevention of inflammatoryand/or hyperproliferative and pruritic skin diseases such as atopicdermatitis, psoriasis, pustular psoriasis, rosacea, keloids,hypertrophic scars, acne, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly aswell as other diseases with epithelial barrier dysfunction such as agedskin.

Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc, wasisolated also from Microcystis. Nostopeptin BN920 inhibited chymotrypsinwith an IC50 value of 31 nM (see J. Nat. Prod. 68(9), 1324-7 (2005)).

These compounds can be produced by fermentation (using chondromycescroactus, myxobacteria) along with other depsipeptides comprising theso-called ahp-substructure (ahp: 3-amino-6-hydroxy-piperidin-2-one) andthe corresponding dehydro-ahp substructure (dehydro-ahp:3-amino-3,4-dihydro-1H-pyridin-2-one), also called “dehydrate” herein,respectively. Therefore, the yield of fermentation with regard to anysingle of these compounds is rather low.

The present invention relates to processes or methods that allowobtaining such cyclic depsipeptides with increased yield and/or in goodpurity.

In view of the many risks, such as epimerization, tautomerization andthe like in the synthesis of a complex molecule with many possibleisomers, it has been possible to find a manufacturing process,preferably comprising a mixture of solid phase peptide synthesis andreactions in solution, that allows to produce cyclic depsipeptides offormula I in good yield and/or the required stereoisomerical purity,especially both. It is possible to reduce the amount of by-products, andeven to improve yield, by converting such by-products, especially thedehydro-ahp substructure and/or an analogue of the desiredahp-comprising products with a five-membered ring instead of the ahp,into the desired final products. This allows to further increase yield.No synthesis has so far come to our attention making use of solid phasepeptide synthesis in this field.

-   (i/a) In a first embodiment, the invention relates to a method or    process for the preparation of a cyclic depsipeptide compound of the    formula I,

-   especially of the formula IA

-   wherein-   A₁ is a bivalent moiety of an amino acid with a terminal carboxy or    carbamoyl group, especially asparagine or glutamine, and is bound at    its right hand side in formula I (corresponding to the C-terminus)    via a carbonyl (preferably the carbonyl of an α-carboxyl group    thereof) to the rest of the molecule; or is C₁₋₈-alkanoyl or    phosphorylated hydroxy-C₁₋₈-alkanoyl;-   X is bound via an N of A₁ and is acyl, or is absent if A₁ is    C₁₋₈-alkanoyl or phosphorylated hydroxy-C₁₋₈-alkanoyl;-   R₂ is C₁₋₈-alkyl, especially methyl;-   R₃ is the side chain of an amino acid, especially of leucine,    isoleucine or valine;-   R₅ is the side chain of an amino acid, preferably of phenylalanine,    leucine, isoleucine or valine;-   R₆ is the side chain of a hydroxy amino acid, especially of    tyrosine;-   R₇ is the side chain of an amino acid, preferably of the amino acid    leucine, isoleucine or valine; and-   Y is hydrogen or C₁₋₈-alkyl;-   or a salt thereof,-   said method comprising-   selectively deprotecting a compound of the formula II

-   especially of the formula IIA

-   wherein Prot is a protecting group, Y is as defined for a compound    of the formula I and X*, A₁*, R₂*, R₃*, R₅*, R₆*, and R₇* correspond    to X, A₁, R₂, R₃, R₅, R₆, and R₇ in formula I, respectively, but    with the proviso that reactive functional groups on these moieties    (such as amino, imino, hydroxy, carboxy, sulfhydryl, amidino,    guanidino, O-phosphono (—O—P(═O)(OH)₂) are present in protected form    at least if they could participate in undesired side reactions, to    result in a compound of the formula III,

-   especially of the formula IIIA,

-   wherein X*, A₁*, R₂*, R₃*, R₅*, R₆*, and R₇* have the meanings just    defined,-   reacting the free hydroxyl group under oxidizing conditions to form    a compound of the formula IV

-   especially IVA,

-   and removing remaining protecting groups to yield a compound of the    formula I, or a salt thereof,-   and, if desired, converting a free compound of the formula I, or    especially IA, into a salt, a salt of a compound of the formula I    into a different salt of a compound of the formula I, or especially    IA, or into the free compound of the formula I, or especially IA,    and/or converting a dehydrate analogue and/or five ring analogue of    a compound of the formula I, or especially IA, into the    corresponding compound of the formula I, or especially IA.

Suitable oxidizing conditions for the oxidation of a compound of theformula III or especially IIIA are usually using IBX in DMSO (J. Org.Chem. 1995, 60, 7272-7276); Pyridinium dichromate or Pyridiniumchlorochromate (Tetrahedron Lett. 1979, 5, 399-402); oxalyl chloride,dimethyl sulfoxide and a tertiary amine (J. Peptide Sci. 2006, 12,140-146), oxoammonium salts (J. Org. Chem. 1985, 50, 1332-1334); alkalihypochlorites catalyzed by oxoammonium salts (J. Org. Chem. 1989, 54,2970-2972); oxoaminium salts (Tetrahedron Lett. 1988, 29, 5671-5672),RuCl₂(PPh₃)₃ (Tetrahedron Lett. 1981, 22, 1605-1608); TEMPO (1 mol %) inthe presence of sodium hypochlorite (Tetrahedron Lett. 1990, 31,2177-2180); NaIO₄, TEMPO, NaBr (Tetrahedron 2006, 62, 8928-8932); SiO₂supported vanadium(IV)oxide and t-BuOOH (Advanced Synthesis & Catalysis2007, 349, 846-848). Preferably, the reaction is performed with IBX inDMSO or preferably in an inert solvent, such as tetrahydrofuran, in thepresence of DMSO, at a temperature between 0-50° C., preferably between20-25° C.

-   (ii/a) A further embodiment of the invention refers to the method or    process described above, in addition comprising manufacturing the    compound of the formula IV or especially IVA by a combination of    Solid Phase Peptide Synthesis (especially For synthesis of the    precursor XX or especially XXA given below for the oligopeptide    precursor of the formula VIII or especially VIIIA given below, or of    the oligopeptide precursor of the formula XXIV or especially XXIVA    given below for the oligopeptide precursor of the formula XXV or    especially XXVA given below) and Solution Phase synthesis    (especially from the compounds just mentioned to the final product)    from the corresponding starting amino acids and side chain    precursors.-   (iii/a) Yet a further embodiment of the invention relates to a    method or process as described above, further comprising, for the    synthesis of a compound of the formula II above, reacting a compound    of the formula VI,

-   especially VIA,

-   wherein Prot is a protecting group, Y is as defined for a compound    of the formula I and R₂*, R₃*, R₅*, R₆*, and R₇* are as defined for    a compound of the formula II above, with an acid of the formula VII

-   or a reactive derivative thereof,-   wherein X** is an amino protecting group or is X*, and wherein X*    and A₁* are as defined for a compound of the formula II above; and,    if X** is an amino protecting group, removing said amino protecting    group X** to yield the derivative of formula II (especially IIA)    wherein, instead of X*, H (a hydrogen) is present and coupling the    resulting amino group with an acyl group X* using the corresponding    acid X*—OH wherein X* is as defined for a compound of the formula II    defined above, or a reactive derivative thereof.-   (iv/a) Another embodiment of the invention relates to the methods or    processes described above, especially in the preceding paragraph,    further comprising cyclization under lactamization of a linear, that    is, not yet cyclic, precursor peptide of the compound of the formula    VI, carrying an N-terminal amino group and a C-terminal carboxy    group, under reaction conditions that allow for the formation of an    amide bond from said amino and said carboxy group, preferably using    Solution Phase chemistry.

Lactamizations in solutions are usually carried out at very lowconcentrations of the substrate in order to avoid oligomerizations andpolymerizations. This requires huge amounts of solvents and very largereactors to carry out the reactions. For example, the macrolactamizationof an oligopeptide is performed at a concentration of 2 mMols/liter inreference Yokokawa et al., Tetrahedron 2005, 61, 1459-1480. Thisdifficulty can be circumvented by dissolving the tertiary base and thecoupling reagent and, in a controlled way adding a solution of theoligopeptide to this solution. The controlled, especially slow, additionof the oligopeptide-solution generates permanently low concentrations ofthe activated oligopeptide in solution and thus prevents oligomerizationand polymerization. The addition rate of the oligopeptide solution canbe adjusted according to the reaction rate for the macrocyclization: ifthe macrocyclization is a fast reaction, the solution of theoligopeptide can be added fast. If the macrocyclization is slow, theaddition of the solution must be slow to ensure permanent lowconcentration of the activated oligopeptide. Thus the controlledaddition of the oligopeptide enables to work with much less solventamounts and still maintaining the concentration of the activatedoligopeptide below 10⁻³ mM, e.g. in the range from 10⁻⁴ to 10⁻⁶ mM oreven lower. This variant of controlled addition of the oligopeptide tothe coupling reagent solution is an embodiment of the invention.

-   (v/a) In yet a further embodiment, the invention relates to the    method or process as described above, especially in the preceding    paragraph, where the linear (this term where used meaning not yet    cyclic) precursor peptide is of the formula VIII,

-   especially VIIIA

-   wherein Prot* is a protecting group that can be cleaved off    selectively without affecting other protecting groups present and is    stable during deprotection steps during synthesis of the linear    precursor peptide (e.g. allyloxycarbonyl) and R₂*, R₃*, R₅*, R₆*,    and R₇* are as defined for a compound of the formula VI as described    above, further comprising, after cyclisation of the compound of the    formula VIII, especially VIIIA, removing the protecting group Prot*    in situ to yield the compound of the formula VI, especially VIA.-   (vi/a) In another embodiment, the invention relates to the method or    process described above, especially in the preceding paragraph,    where the linear precursor peptide of the formula VIII, especially    VIIIA, is synthesized from the corresponding amino acids by solid    phase peptide synthesis and subsequent cleavage from the employed    solid support.-   (vii/a) An embodiment of the invention further relates to the method    or process as described above (especially in the preceding paragraph    (vi/a)), further comprising either in a variant a), coupling an    amino acid of the formula IX,

-   especially of the formula IXA,

-   wherein R₃* is as defined for a compound of the formula II above and    Prot** is an amino protecting group that can be removed on the resin    without cleaving other bonds, or a reactive derivative of said amino    acid, via an oxygen to a cleavable linker L which is bound to a    solid resin RES (e.g. by reaction with a resin of the formula    (X−L)_(z)-RES wherein L and RES are as just defined, X is e.g. halo,    e.g. chloro, and z is a number larger than zero, e.g. a natural    number)-   and removing the protecting group Prot**;-   coupling the obtainable resin bound amino acid symbolized by the    formula X,

-   especially XA,

-   in which RES and R₃* are as defined for a compound of the formula    IX, n is a (e.g. natural) number larger than zero and L is a    cleavable linker, with an amino acid of the formula XI,

-   especially XIA,

-   wherein Prot* is as defined for a compound of the formula VIII above    and R₂* is as defined for a compound of the formula II above, or a    reactive derivative of said amino acid, coupling the obtainable    resin bound dipeptide symbolized by the formula XII,

-   especially XIIA;

-   in which Prot* is as defined for a compound of the formula VIII    above, R₂* and R₃* are as defined for a compound of the formula II    above, and n, L and RES are as defined for a compound of the formula    X, via the free hydroxy group with an amino acid of the formula    XIII,

-   especially XIIIA,

-   wherein Prot** is as defined for a compound of the formula IX and    R₇* is as defined for a compound of the formula II above, or a    reactive derivative of said amino acid, and removing the protecting    group Prot**;-   or, in a variant b), coupling a dipeptide of the formula XXVII,

-   especially of the formula XXVIIA,

-   wherein R₃* and Prot** are as described for a compound of the    formula IX, especially IXA, and Prot* is as defined for a compound    of the formula VIII above, or a reactive derivative of said    dipeptide, to an amino acyl moiety, bound via an oxygen to a    cleavable linker L which is bound to a solid resin RES, having the    formula X,

-   especially XA,

-   that can be obtained as described under variant a), in which RES,    and R₃* are as defined for a compound of the formula IX and L and    RES are as just defined;-   and removing the protecting group Prot**;-   and, after the reactions of variant a) or of variant b),-   (viii/a) coupling the obtainable compound of the formula XIV,

-   especially XIVA,

-   wherein R₂*, R₃* and R₇* are as defined for a compound of the    formula II above, Prot* is as defined for a compound of the formula    VIII above and n, L and RES are as defined for a compound of the    formula X, with an amino acid of the formula XV,

-   especially the formula XVA,

-   in which R₆* and Y are as defined for a compound of the formula II    above and Prot** is as defined for a compound of the formula IX    above, or a reactive derivative of said amino acid, and removing the    protecting group Prot**;-   (ix/a) preferably coupling the obtainable compound of the formula    XVI

-   especially the formula XVIA,

-   wherein Y, R₂*, R₃*, R₇* and R₆* are as defined for a compound of    the formula II above, Prot* is as defined for a compound of the    formula VIII above and n, L and RES are as defined for a compound of    the formula X, with an amino acid of the formula XVII,

-   especially formula XVIIA,

-   wherein R₅* is as defined for a compound of the formula II above and    Prot** is as defined for a compound of the formula IX, or a reactive    derivative of said amino acid, and removing the protecting group    Prot**,-   and preferably-   (x/a) finally coupling the resulting compound of the formula XVIII,

-   especially XVIIIA,

-   wherein Y, R₂*, R₃*, R₇*, R₆* and R₅* are as defined for a compound    of the formula II above, Prot* is as defined for a compound of the    formula VIII above and n, L and RES are as defined for a compound of    the formula X above, to an unnatural amino acid (=synthon) of the    formula XIX,

-   especially the formula XIXA,

-   wherein Prot is as defined for a compound of the formula II above    and Prot** is as defined for a compound of the formula IX, or an    activated derivative of said synthon, and removing the protecting    group Prot** to yield a compound of the formula XX,

-   especially XXA,

-   wherein Prot, Y, R₂*, R₃*, R₇*, R₆* and R₅* are as defined for a    compound of the formula II above, Prot* is as defined for a compound    of the formula VIII above and n, L and RES are as defined for a    compound of the formula X,-   and-   (xi/a) cleaving the solid phase bound peptide in formula XX off the    solid phase L-RES to yield the corresponding compound of the formula    VIII, especially VIIIA, as shown above.

Another embodiment of the invention relates to the synthesis of acompound of the formula II as given above according to section (i/a),preferably preceded by the reaction according to section (ii/a) or morepreferably according to section (iii/a); preferably preceded by thereaction according to section (iv/a) or preferably (via), preferablypreceded by the reaction according to section (vi/a), preferablypreceded by the reaction according to section (x/a), preferably precededby the reaction according to section (ix/a), preferably preceded by thereaction according to section (viii/a), preferably preceded by thereaction according to section (vii/a).

-   (i/b) Another embodiment of the invention relates to a method or    process above, comprising, for the synthesis of the compound of the    formula II given above, cyclization under lactamization of a linear,    not yet cyclic, precursor peptide of the compound of the formula II,    carrying an N-terminal amino group and a C-terminal carboxy group,    under reaction conditions that allow for the formation of an amide    bond from said amino and said carboxy group, preferably using    Solution Phase chemistry.-   (ii/b) A further embodiment of the invention relates to the method    or process according to the preceding paragraph (i/b), where the    linear precursor peptide is of the formula XXV,

-   especially XXVA,

-   wherein X*, A₁*, R₂*, R₃*, R₅*, R₆*, R₇* and Prot are as defined for    a compound of the formula II above.-   (iii/b) Another embodiment refers to the method or process according    to the preceding paragraph (ii/b), further comprising, for the    synthesis of the compound of the formula XXV, cleaving a compound of    the formula XXIV,

-   especially XXIVA,

-   wherein X*, A₁*, R₂*, R₃*, R₅*, R₆*, R₇* and Prot are as defined for    a compound of the formula II above, L is a cleavable linker, RES is    a solid resin, n is a natural number and Prot** is an amino    protecting group that can be removed without parallel removal of the    protecting group Prot and with the product remaining on the resin,    and (before the cleavage, in parallel or subsequently to it)    removing the protecting group Prot** to yield the compound of the    formula XXV.-   (iv/b) A further embodiment of the invention relates to the method    or process according to the preceding paragraph (iii/b), further    comprising, for the synthesis of the compound of the formula XXIV,    coupling an amino acid of the formula XIX,

-   especially XIXA,

-   wherein Prot is as defined for a compound of the formula II above    and Prot** is as defined for a compound of the formula XXIV above,    or an activated derivative of said amino acid, with a compound of    the formula XXIII,

-   especially XXIIIA,

-   wherein X*, A₁*, R₂*, R₃*, R₅*, R₆* and R₇* are as defined for a    compound of the formula II above, L is a cleavable linker, RES is a    solid resin, and n is a natural number.-   (v/b) Yet a further embodiment of the invention relates to the    method or process according to the preceding paragraph (iv/b),    further comprising, for the synthesis of the compound of the formula    XXIII, coupling an amino acid of the formula XVII*

-   especially XVIIA*,

-   wherein R₅* is as defined for a compound of the formula II above and    Prot*** is an amino protecting group that can be cleaved off    selectively without affecting other protecting groups present and    with the product remaining on the resin, or a reactive derivative of    said amino acid, with a compound of the formula XXII,

-   especially XXIIA

-   wherein X*, A₁*, R₂*, R₃*, R₆* and R₇* are as defined for a compound    of the formula II above, L is a cleavable linker, RES is a solid    resin, and n is a natural number, and removing the protecting group    Prot***.-   (vi/b) In yet a further embodiment, the invention relates to the    method or process according to the preceding paragraph (v/b),    further comprising, for the synthesis of the compound of the formula    XXII, coupling an amino acid of the formula XV*,

-   especially XVA*

-   in which R₆* and Y are as defined for a compound of the formula II    above and Prot*** is an amino protecting group that can be cleaved    off selectively without affecting other protecting groups present    and with the product remaining on the resin, or a reactive    derivative of said amino acid, with a compound of the formula XXI,

-   especially XXIA,

-   wherein X*, A₁*, R₂*, R₃* and R₇* are as defined for a compound of    the formula II above, L is a cleavable linker, RES is a solid resin,    and n is a natural number, and removing the protecting group    Prot***.-   (vii/b) Another embodiment of the invention relates to the method or    process according to the preceding paragraph (vi/b), further    comprising, for the synthesis of a compound of the formula XXI,    reacting an amino acid of the formula XIII*,

-   especially XIIIA*,

-   wherein Prot*** is an amino protecting group that can be cleaved off    selectively without affecting other protecting groups present and    with the product remaining on the resin, and R₇* is as defined for a    compound of the formula II above, or a reactive derivative of said    amino acid,-   with the hydroxyl group of a compound of the formula XXVI,

-   especially XXVIA,

-   wherein X*, A₁*, R₂* and R₃* are as defined for a compound of the    formula II above, L is a cleavable linker, RES is a solid resin, and    n is a natural number;-   and removing the protecting group Prot***.-   (viii/b) In a further embodiment, the invention relates to the    method or process according to the preceding paragraph (vii/b),    further comprising, for the synthesis of a compound of the formula    XXVI, especially XXVIA, coupling a resin bound dipeptide symbolized    by the formula XII*,

-   especially XIIA*

-   in which Prot**** is a protecting group that can be cleaved off    selectively without affecting other protecting groups present in a    compound of the formula II as defined above and with the product    remaining on the resin, R₂* and R₃* are as defined for a compound of    the formula II above, L is a cleavable linker, RES is a solid resin,    and n is a natural number, after removal of the protecting group    Prot**** via the thus obtainable free amino group, with. an acid of    the formula VII

-   wherein X** is an amino protecting group or is X*, and wherein X*    and A₁* are as defined for a compound of the formula II above, or a    reactive derivative of said acid;-   and, if X** is an amino protecting group, removing said amino    protecting group X** to yield the derivative of formula II wherein,    instead of X*, H is present and coupling the resulting amino group    with an acyl group X* using the corresponding acid X*—OH wherein X*    is as defined for a compound of the formula II above, or a reactive    derivative of said acid.-   (ix/b) A yet further embodiment of the invention relates to the    method or process according to the preceding paragraph (viii/b),    further comprising, for the synthesis of a compound of the formula    XII, coupling a resin bound amino acid symbolized by the formula X,

-   especially XA,

-   wherein R₃* is as defined for a compound of the formula II above, L    is a cleavable linker, RES is a solid resin, and n is a natural    number,-   with an amino acid of the formula XI*,

-   especially XIA*,

-   wherein Prot**** is a protecting group that can be cleaved off    selectively without affecting other protecting groups present and    with the product remaining on the resin, and R₂* is as defined for a    compound of the formula II above, or a reactive derivative of said    amino acid.-   (x/b) A further embodiment of the invention relates to the method or    process according to the preceding paragraph (ix/b), further    comprising, for obtaining the resin bound amino acid of the formula    X, coupling an amino acid of the formula IX*,

-   especially IXA*,

-   wherein R₃* is as defined for a compound of the formula II in claim    1 and Prot*** is an amino protecting group can be cleaved off    selectively without affecting other protecting groups present and    with the product remaining on the resin; or a reactive derivative of    said amino acid of the formula IX, to a cleavable linker L which is    bound to a solid resin RES, and removing the protecting group    Prot***.-   (i/c) Another embodiment of the invention relates to the method or    process according to any one of the preceding paragraphs (i/a) to    (x/b) where the symbols A₁, R2, R3, R5, R6, R7, X and Y or the    corresponding unprotected or protected moieties R₂*, R₃*, R₅*, R₆*,    R₇*, X* and Y3 are selected so that, in the resulting compound of    the formula I, or a salt thereof,-   A₁ is the bivalent radical of L-glutamine bound via the carbonyl of    its α-carboxy group to the amino group at the right of A₁ in formula    I and via its α-amino group to X, or is    2S-(2-hydroxy-3-phosphonooxy)-propionyl;-   R₂ is methyl;-   R₃ is isopropyl, isobutyl (2-methyl-n-propyl wherever used) or    benzyl, especially isobutyl;-   R₅ is sec-butyl or benzyl, especially sec-butyl;-   R₆ is 4-hydroxybenzyl;-   R₇ is isopropyl or sec-butyl (1-methyl-n-propyl wherever used),    especially sec-butyl;-   X is acetyl or isobutyryl, or is absent if A₁ is    2S-(2-hydroxy-3-phosphonooxy)-propionyl and-   Y is methyl. This paragraph is also named paragraph a) below.-   (i/d) In another particular embodiment, the invention relates to a    method or process for converting a dehydrate of a compound of the    formula I given above or in particular with the substituents as    defined in the preceding paragraph (i/c) into the corresponding    compound of the formula I, where the dehydrate has the formula V,

-   especially VA,

-   in which Y, X, A₁, R₂, R₃, R₅, R₆ and R₇ are as defined for a    compound of the formula I above;-   or especially a method or process for shifting the equilibrium of a    mixture of a compound of the formula I and its corresponding    dehydrate, and/or its corresponding hemiaminal analogue with a    five-ring instead of the ahp structure in formula I which may also    be formed as byproduct and has the formula V*,

-   especially the formula VA*,

-   in which Y, X, A₁, R₂, R₃, R₅, R₆ and R₇ are as defined for a    compound of the formula I above, respectively;-   in favor of the compound of the formula I,-   said method or process comprising using an aqueous acid as reactive    solvent to drive the reaction. This method can be used independently    (e.g. also for the product of fermentation or biosynthesis) or in    addition to the other processes or methods described above and below    to increase the yield or to re-convert a compound of the formula V,    especially VA, and/or the analogue with a five-membered ring instead    of the ahp structure in formula I, into the corresponding compound    of the formula I.

The method described for the conversion of the dehydrate and/or the fivering analogue (always regarding the desired ahp ring) into the desiredcompound of the formula I or especially IA, e.g. of Compound A-dehydratefrom Example 3 B into Compound A, enables a straight-forward synthesisof this class of compounds. Up to now, an acidic treatment as final stephad to be circumvented in order to avoid the dehydration of the product.

-   (i/e) A further embodiment of the invention relates to the method    according to the preceding paragraph (i/d), wherein the acid is a    carboxylic acid, especially a halo substituted C₁₋₈alkanoic acid,    more especially trifluoroacetic acid or trichloroacetic acid.-   (i/f) The invention, in yet a further embodiment, relates to a    compound of the formula II,

-   wherein Prot is a protecting group, Y is as defined for a compound    of the formula I in the first instance above or in particular as or    claim 17 and X*, A₁*, R₂*, R₃*, R₅*, R₆*, and R₇* correspond to X,    A₁, R₂, R₃, R₅, R₆, and R₇ in formula I as defined in claim 1 or in    paragraph (ia) given above, respectively, however with the proviso    that reactive functional groups on these moieties are present in    protected form.-   (i/g) In a further embodiment, the invention relates to a novel    compound selected from the group consisting of compounds of the    formula II, III, IV, V, VI, VIII, X, XII, XIV, XVI, XVIII, XIX, XX,    XXI, XXII, XXIII, XXIV, XXV, XXVI and XXVIII, and especially of the    formula IIA, IIIA, IVA, VA, VIA, VIIIA, XA, XIIA, XIVA, XVIA,    XVIIIA, XIXA, XXA, XXIA, XXIIA, XXIIIA, XXIVA, XXVA, XXVIA and    XXVIIIA, yet more especially to the group consisting of the    following compounds given in the examples: From Scheme 1: compound    2, compound 3, compound 4, synthon 1; from Scheme 2: compound 5;    Fmoc-Leu-Linker-Resin according to Example 1B(2);    Fmoc-Thr-Leu-Linker-Resin according to Example 1B(3);    Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin according to Example 1B(4);    Isobutyryl-Gln(Trt)-Thr-Leu-Linker-Resin according to Example 1B(5);    Isobutyryl-Gln(Trt)-Thr(Ile-Fmoc)-Leu-Linker-Resin according to    Example 1B(6); the product of Example    1B(7)=Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Fmoc)-Leu-Linker-Resin    (previously named:    Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Fmoc)-Leu-Linker-Resin);    Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Fmoc)-Leu-Linker-Resin    (previously named:    Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Ile-Fmoc)-Leu-Linker-Resin)    according to Example 1B(8);    Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon    1-H)-Leu-Linker-Resin (previously named:    Isobutyryl-Gln(Trt)-Thr(Ile-N-Me-Tyr(tBu)-Ile-Synthon    1-H)-Leu-Linker-Resin) according to example 1B(9).    Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-OH    (previously named:    Isobutyryl-Gln(Trt)-Thr(Ile-N-Me-Tyr(tBu)-Ile-Synthon 1-H)-Leu-OH)    according to example 1B(10) and Scheme 3; H-Thr-Leu-Resin according    to example 1B(12); H-Gln(Trt)-Thr-Leu-Resin according to example    1B(13); Isobutyryl-Gln(Trt)-Thr-Leu-Resin according to example    1B(14); Isobutyryl-Gln(Trt)-Thr(Ile-H)-Leu-Resin according to    example 1B(15); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-H)-Leu-Resin    according to example 1B(16);    Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-H)-Leu-Resin according to    example 1B(17); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon    1-H)-Leu-Resin according to example 1B(18); from Scheme 3, Compound    6 and/or 7 (the latter preferred); from Scheme 4: Compound 8 and the    5-ring hemiaminal-isomer; from Scheme 5, precursor peptide 2,    compound 9, compound 10 (this one being preferred in the present    enumeration regarding Scheme 5), and/or compound 11;    Fmoc-Thr-Leu-Trt-Tentagel-S from Example 2A(1);    Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S according to Example 2A(2);    Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S according to Example 2A(3);    Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S according to Example    2A(4); Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Fmoc)-Leu-Trt-Tentagel-S    (previously named:    Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Fmoc)-Leu-Trt-Tentagel-S)    according to Example 2A(5);    Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Fmoc)-Leu-Trt-Tentagel-S    (previously named:    Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Phe-Fmoc)-Leu-Trt-Tentagel-S)    according to Example 2A(6); and    Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Synthon1-H)-Leu-OH (previously    named: Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Phe-Synthon1-H)-Leu-OH)    (precursor peptide 2) according to Example 2A(7).

The following definitions (or also definitions already included above)can replace more general terms used in invention embodiments above andbelow in order to define further embodiments of the invention, witheither one, two or more or all general terms being replaceable by themore specific terms in order to define such invention embodiments:

A bivalent moiety of an amino acid with a terminal carboxy or carbamoylgroup is preferably an alpha-carbamoyl or carboxyl-C₁₋₈-substitutedamino acid, especially the bivalent moiety of asparagine or glutamine,and is bound at its right hand side in formula I via a carbonyl(preferably the carbonyl of its α-carboxyl group) to the rest of themolecule.

C₁₋₈-alkanoyl or phosphorylated hydroxy-C₁₋₈-alkanoyl (C₁₋₈-alkanoylcarrying both a hydroxyl and a phosphono (—O—P(═O)(OH)₂) group) A₁ ise.g. 2,3-dihydroxy-propanoyl (preferably in S-form) or2-hydroxy-3-phosphono-propanoyl (preferably in S-form).

R₂ and R₂* are C₁₋₈-alkyl, especially methyl wherever mentioned.

R₃ is the side chain of an amino acid, especially of a natural aminoacid. Preferably, it is C₁₋₈ alkyl which may be branched or linear. Mostespecially, C₁₋₈alkyl is n-(2-methyl)propyl(isobutyl),n-(1-methylpropyl(sec-butyl) or methyl, that is, the amino acid carryingthe moiety is leucine, isoleucine or valine.

R₃* is the corresponding side chain in protected form if a functionalgroup is present that has to be hindered to participate in a reaction.Preferably, it is C₁₋₈alkyl which may be branched or linear, especiallyas defined in the preceding paragraph.

A “side chain of an amino acid” may be selected from any moiety, e.g. amono- or polycyclic, linear, saturated, unsaturated (e.g. withconjugated double bonds) or partially saturated organic moiety, e.g.with up to 20 carbon atoms and 0 to 5 heteroatoms in the basis structureindependently selected from N, O and S replacing the correspondingnumber of carbon atoms, and may be substituted by up to three moietiesselected from amino, imino, hydroxy, carboxy, carbamoyl, sulfhydryl,amidino, guanidino, O-phosphono (—O—P(═O)(OH)₂). Preferably, the sidechains are selected from those of the 20 standard alpha-amino acidsarginine, histidine, lysine, aspartic acid, glutamic acid, serine,threonine, asparagine, glutamine, cysteine, glycine, alanine, leucine,isoleucine, methionine, phenylalanine, tryptophan, tyrosine, valine andfurther proline (then with internal cyclization including thealpha-amino group).

For the amino acids, either their names or the customary three lettercodes are used in the present disclosure, in accordance with thefollowing table:

Amino acid Three letter code Alanine Ala Arginine Arg Asparagine AsnAspartic acid Asp Asparagine or aspartic acid Asx Cysteine Cys Glutamicacid Glu Glutamine Gln Glutamine or glutamic acid Glx Glycine GlyHistidine His isoleucine Ile Leucine Leu Lysine Lys Methionine MetPhenylalanine Phe Proline Pro Serine Ser Threonine Thr Tryptophan TryTyrosine Tyr Valine Val

R₅ is the side chain of an amino acid, preferably a standard amino acid.Preferably, it is C₁₋₈ alkyl which may be branched or linear and whichis unsubstituted or substituted by phenyl. Most especially it is benzyl,n-(2-methyl)propyl, isobutyl or methyl, that is, the amino acid carryingthe moiety is phenylalanine, leucine, isoleucine or valine.

R₆ is the side chain of a hydroxy amino acid, especially of tyrosine.

R₇ is the side chain of an amino acid, especially of a natural aminoacid. Preferably, it is C₁₋₈alkyl which may be branched or linear. Mostespecially it is n-(2-methyl)propyl(isobutyl),n-(1-methyl)propyl(sec-butyl) or methyl, that is, the amino acidcarrying the moiety is leucine, isoleucine or valine.

C₁₋₈-alkyl can be linear or branched one or more times; for example, itcan be n-(2-methyl)propyl, n-(1-methyl)propyl or methyl.

All of the compounds can, where salt-forming groups such as basicgroups, e.g. amino or imino, or acidic groups, e.g. carboxyl or phenolichydroxyl, are present, be used in free form or as salts or as mixturesof salts and free forms. Thus where ever a compound is mentioned, thisincludes all these variants. For example, basic groups may form saltswith acids, such as hydrohalic acids, e.g. HCl, sulfuric acid or organicacids, such as acetic acid, while acidic groups may form salts withpositive ions, e.g. ammonium, alkylammonium, alkali or alkaline-earthmetal salt cations, e.g. Ca, Mg, Na, K or Li cations, or the like.

“Or the like” or “and the like”, wherever used in this disclosure,refers to the fact that other alternatives to those mentioned precedingsuch expression are known to the person skilled in the art and may beadded to those expressions specifically mentioned; in other embodiments,“or the like” and “and the like” may be deleted in one or more or allinvention embodiments.

The protecting groups Prot, Prot*, Prot**, Prot***, Prot**** and anyfurther protecting groups present on the moieties A*, R₂*, R₃*, R₅*,R₆*, R₇*, X*, where ever mentioned throughout the present descriptionand claims, are selected so that they allow for orthogonal protection.

Orthogonal protection is a strategy allowing the deprotection ofmultiple protective groups one (or more but not all) at the time wheredesired each with a dedicated set of reaction conditions withoutaffecting the other protecting group(s) or bonds to resins, e.g. vialinkers on solid synthesis resins. In other terms: The strategy usesdifferent classes of protecting groups that are removed by differentchemical mechanisms, also using appropriate linkers in the case of solidphase peptide synthesis (where the linker-resin bond might together beconsidered as a carboxy protecting group).

Preferably, the protecting groups are selected as follows:

The protecting group Prot is preferably selected so as to resist removalof any other protecting groups used or present during the synthesisaccording to the invention of a depsipeptide, e.g. able to resist mildbases (see Prot*), but removable with fluoride ion (especially underanhydrous conditions), e.g. Bu₄N⁺F⁻ (also if created in situ, e.g. usingBu₄N⁺Cl⁻ with KF.H₂O, KF with 18-crown-6, LiBr with 18-crown-6,BF₃.diethylether, pyridine-HF, HF in urea, Et₃N(HF)₃ (wherein Et isethyl) or the like, where the solvent is e.g. selected from the groupconsisting of N,N-dimethylformamide, acetonitrile, chloroform andtetrahydrofurane.

Preferably, Prot is an ether protecting group, especially selected fromthe group consisting of silyl protecting groups in which the silylmoiety carries up to three organic moieties bound via a carbon(optionally via a further Si atom), such as tert-butyldiphenylsilyl,trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl,triphenylsilyl, diphenylmethylsilyl, ti-tert-butyldimethylsilyl,tert-butylmethoxyphenylsilyl, tris(trimethylsilyl)silyl or the like.

Prot* is a protecting group that can be cleaved off selectively withoutaffecting other protecting groups present, and also without affectingthe depsipeptide forming ester bond or a linker to a resin RES, and isstable during deprotection steps during synthesis of the linearprecursor peptide (e.g. removal of allyloxycarbonyl); it is preferably aprotecting group removable by specific triphenylphosphin complexes inthe presence of metal hydrides or other reductants, e.g. (PH₃P)₄Pdpreferably in combination with di-n-butyl tin hydride or tri-n-butyl tinhydride, phenylsilane, sodium borohydride or dimedone, in an appropriatesolvent, e.g. tetrahydrofurane, and is preferably not cleavable underconditions that allow for the removal of a protecting group Prot**; forexample, and Prot* is selected from the group consisting ofC₃-C₈alk-2-enyloxycarbonyl moieties, e.g. allyloxycarbonyl (Alloc),1-isopropylallyloxycarbonyl, 4-nitrocinnamyloxycarbonyl and3-(3′-pyridyl)prop-2-enyloxycarbonyl.

Prot** is a protecting group that can be removed on the resin withoutcleaving other bonds (no cleavage of an amino acid or peptide bound viathe carbonyl of its (especially α-carboxyl group to the binding via alinker L mentioned below; also without cleaving off the protecting groupProt once present), especially a protecting group removable withoutcleavage of an ester (instead of an amide) bond in a depsipeptide ordepsipeptide precursor and under conditions other than those for theprotecting groups Prot* and Prot, while preserving the binding via thelinker to a resin RES where present; it is preferably removable by amild base e.g. piperidine, morpholine, dicyclohexylamine,p-dimethyl-amino-pyridine, diisopropylamine, piperazine,tris-(2-aminoethyl)amine in an appropriate solvent, e.g.N,N-dimethylformamide, methylene chloride; Prot** is, e.g., selectedfrom the group consisting of fluoren-9-ylmethoxycarbonyl (Fmoc); 2-(2′or 4′-pyridyl)ethoxycarbonyl and 2,2-bis(4′nitrophenyl)ethoxycarbonyl.

Prot*** (an amino protecting group that can be cleaved off selectivelywithout affecting other protecting groups present and with the productremaining on the resin) and Prot**** (a protecting group that can becleaved off selectively without affecting other protecting groupspresent in a compound of the formula II as defined above and below andwith the product remaining on the resin) are preferably protectinggroups that can be removed as Prot** and are, e.g., selected from thosementioned for R**, e.g. fluoren-9-ylmethoxycarbonyl (Fmoc).

The preferred orthogonal synthesis method in this case makes use of theFmoc method known in general for peptide synthesis using solid phasepeptide synthesis combined with solution phase macrolactamization andfurther chemical conversions.

Alternatively, e.g. the Boc protecting group might be used instead ofFmoc Prot**, Prot*** and Prot****.

However, this will require different side chain protecting groups andalso the hydroxy-group of N-methyl-Tyr will then have to be protected ina different way to maintain orthogonality of the protection groups.

Other protecting groups present as well as the binding linker to a resinRES where present are preferably not removable under conditions underwhich Prot* and Prot** can be removed, e.g. in A*, the amide can beN-protected e.g. with trityl (triphenylmethyl) (cleavage e.g. withtrifluoro acetic acid (TFA); in R₆* a tyrosine hydroxy can be protectedas t-butyl-ether, or protected by tert-butyldimethylsilyl,methoxymethyl, Boc (tert-butoxycarbonyl) or arylacetate (cleavage withTFA).

Appropriate protecting groups are known in the art, as well methods fortheir introduction and removal. For example, the protecting groups,their introduction and removal methods may be selected from thosedescribed in standard textbooks such as “Protective Groups in OrganicSynthesis”, 3^(rd) ed., T. W. Green and P. G. M. Wuts (Eds.). J. Wiley &Sons, Inc., New York etc. 1999.

The protecting groups Prot, Prot*, Prot**, Prot***, Prot**** and otherprotecting groups are thus not limited to those mentioned above—ratherthey should fulfill conditions that make them appropriate for orthogonalprotection, e.g. as described above or below.

It is recommended to avoid too basic conditions (though the basesdescribed for Fmoc cleavage, such as piperidine, are usually allowable)to avoid cleavage of the depsipeptide (ester) bond.

Among the possible solid support for Solid Phase Peptide Synthesis(SPPS), the following may be mentioned:

-   -   Gel-type supports without or with spacer: These are highly        solvated polymers with an equal distribution of functional        groups. This type of support is the most common, and includes:

-   Polystyrene: Styrene cross-linked with e.g. 1-2% divinylbenzene;    Polyacrylamide or polymethacrylamide: as hydrophilic alternative to    polystyrene; Polyethylene glycol (PEG): PEG-Polystyrene (PEG-PS) is    more stable than polystyrene and spaces the site of synthesis from    the polymer backbone; PEG-based supports: Composed of a    PEG-polypropylene glycol network or PEG with polyamide or    polystyrene (these already include a spacer, PEG);    -   Surface-type supports: Materials developed for surface        functionalization, including controlled pore glass, cellulose        fibers, and highly cross-linked polystyrene.    -   Composites: Gel-type polymers supported by rigid matrices.

Usually these gels carry reactive groups to which a linker L asmentioned for various precursors above and below can be bound. Forexample, such groups include aminomethyl groups, polyethyleneglycolgroups with a terminal hydroxy, and the like.

Any such support can be used in the embodiments of the presentinvention.

Gel type supports are used in another special embodiment of theinvention, Among these, polystyrene (divinylbenzene crosslinked);polyacrylamide and polymethacrylamide resins are especially preferred.

Among the possible linkers, all commonly known and appropriate may beused.

Examples in possible embodiments of the invention are the2-methoxy-4-benzyloxy-benzyl alcohol linker (a Sasrin-Linker, Sasrinstands for superacid sensitive resin, binds the amino acids or peptidesvia alcoholic OH); the trityl linker family (e.g, Trityl, 2Cl-Trityl,which bind the amino acids or peptides via OH); the4-(2,4-dimethoxyphenylhydroxymethyl)phenoxymethyl-Linker(Rink-Acid-Linker, binds the amino acids or peptides via OH); ortris(alkoxy)benzyl ester linkers (HAL-Linker, binds the amino acids orpeptides via OH).

Where reactive derivatives of acids, especially amino acids, orpeptides, e.g. dipeptides, are mentioned, they may be formed in situ ormay be used as such.

Reactive (or active) derivatives used as such include the acyl-halides,e.g. acyl-chlorides, -fluorides or -nitrophenyl esters, e.g. the2,4-dinitrophenyl esters, or acid anhydrides (symmetric or e.g. withacetic acid) of the carboxy groups of the acids to be reacted.

For in situ amino acid activation, customary coupling agents may beapplied. Such reagents are known to the person skilled in the art andcan be purchased conveniently from many sources, e.g. AldrichChemFiles—Peptide Synthesis (Aldrich Chemical Co., Inc., Sigma-AldrichCorporation, Milwaukee, Wis., USA) Vol. 7 No. 2, 2007 (seehttp://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/Brochure/al_chemfile_v7_n2.Par.0001.File.tmp/al_chemfile_v7_n2.pdf).Among the possible coupling agents for amide and ester bond synthesisthe following may be mentioned:

Triazoles, uronium or hexafluorophosphonium derivatives, e.g.1-hydroxy-benzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt),ethyl 2-cyano-2-(hydroxyimino)acetate,2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate methanaminium (HATU),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate(HBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluoroborate(TBTU), 2-succinimido-1,1,3,3-tetramethyluronium-tetrafluoroborate(TSTU),2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium-tetrafluoroborate(TNTU),O-[(cyano(ethoxycarbonyl)-methyliden)amino]-1,1,3,3-tetramethyluronium-tetrafluoroborate(TOTU), O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethylene uroniumhexafluorophosphate (HBMDU),O-(benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU),O-(benzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uroniumhexafluorophosphate (HBPipU),3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt),1-hydroxy-7-aza-benzotriazole and its corresponding uronium orphosphonium salts, designated HAPyU and AOP,1-cyano-2-ethoxy-2-oxoethylideneaminooxy-dimethylamino-morpholino-carbeniumhexafluorophosphate (COMU), chlorotripyrrolidinophosphoniumhexafluorophosphate (PyCloP), or the like;

Carbodiimides, e.g. dicyclohexylcarbodiimide,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide,1-tert-butyl-3-ethylcarbodiimide,N-cyclohexyl-N′-2-morpholinoethyl)carbodiimide ordiisopropylcarbodiimide (especially for ester formation via O-acyl ureaformation of the carboxylic group); or

-   active ester forming agents, e.g. 2-mercaptobenzothiazole (2-MBT),-   azide forming agents, e.g. diphenyl phosphoryl azide,-   acid anhydrides, such as propane phosphonic acid anhydride,-   acid halogenation agents, e.g.    1-chloro-N,N,2-trimethyl-1-propenylamine,    chloro-N,N,N′,N′-bis(tetramethylene)formamidinium tetrafluoroborate    or hexafluorophosphate, chloro-N,N,N′,N′-tetramethlformamidinium    hexafluorophosphate, fluoro-N,N,N′,N′-tetramethylformamidinium    hexafluorophosphate,    fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium    hexafluorophosphate,-   or the like, or mixtures of two or more such agents.

Also for the ester coupling of compounds of the formula XII or XIIA withthose of the formula XIII or XIIIA, respectively, or of compounds of theformula XIII* or XIIIA*with those of the formula XXVI or XXVIA,respectively, the corresponding reactive carboxyl compounds can be usedor formed in situ. Here, especially MSNT is preferred as coupling agentas this allows for the maintenance of high stereospecificity.

The reaction may, where appropriate, be conducted in the presence of amild base (e.g. N-methylmorpholine, a trialkylamine, e.g.ethyldiisopropylamine, a di-(alkyl)aminopyridine, such asN,N-dimethylaminopyridine, or the like (taking care that the conditionsare not so basic as to allow for the hydrolysis of ester groups, e.g.the depsipeptide ester group, present in precursors of the compound ofthe formula I), where appropriate or required in the presence of anappropriate solvent or solvent mixture, e.g. an N,N dialkyl-formamide,such as dimethylformamide, a halogenated hydrocarbon, e.g.dichloromethane, N-alkylpyrrolidones, such as N-methylpyrrolidone,nitriles, e.g. acetonitrile, or further an aromatic hydrocarbon, e.g.toluene, or mixtures of two or more, where, provided an excess ofcoupling agent is present, also water may be present. The temperaturesmay be ambient temperature or lower or higher, e.g. in the range from−20° C. to 50° C.

The amino acids of the formula IX, IXA, XI, XIA, XIII, XIIIA, XV, XVA,XVII, XVIIA, XXVII (obtainable e.g. by Solution Phase synthesis), XVII*,XVIIA*, XV*, XVA*, XIII*, XIIIA*, XI*, XIA*, IX* and XIA* are known orthey can be synthesized according to methods known in the art, they arecommercially available, and/or they can be synthesized in analogy tomethods known in the art.

Also the remaining starting materials, e.g. the acid of the formula XIXor VII, or the dipeptide of the formula XXVII or XXVIIA, are known orthey can be synthesized according to methods known in the art, they arecommercially available, and/or they can be synthesized in analogy tomethods known in the art.

For example, the synthon of the formula XIX can be prepared as describedin Example 1 A(4) (which is a specific embodiment of the invention) orin analogy thereto. The synthesis of the intermediate compound 1(Scheme 1) is described in Tetrahedron 61, 1459-1480 (2005).

The coupling reactions for dipeptides make use of the correspondingcarboxylic groups of amino acids in free form or in activated form.

EXAMPLES The following examples illustrate the invention withoutlimiting its scope.

Abbreviations

-   aq. aqueous-   Boc/BOC tert-Butoxycarbonyl-   brine sodium chloride solution in water (saturated at RT)-   Bzl benzyl-   COMU    1-cyano-2-ethoxy-2-oxoethylideneaminooxy-dimethylamino-morpholino-carbenium    hexafluorophosphate-   DCM dichloromethane-   DIPEA N,N-diisopropylethylamine-   DMAP 4-Dimethylaminopyridine-   DMF N,N-dimethylformamide-   Fmoc/FMOC 9-fluorenymethoxycarbonyl-   Et ethyl-   HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate Methanaminium-   HFIP Hexafluoroisopropanol-   HPLC High Performance Liquid Chromatography-   HR-MS High Resolution Mass Spectroscopy-   IPA Isopropylacetate-   IPC In-Process Control-   IR Infrared Spectroscopy-   IT internal temperature-   Kaiser test Ninhydrin-based test to monitor deprotection in SPPS    (see E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook,    Analytical Biochemistry 1970, 34 595); if mentioned to be OK, this    means successful deprotection.-   me methyl-   MS Mass Spectroscopy-   MSNT 1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole-   NMR Nuclear Magnetic Resonance Spetroscopy-   PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium    hexafluorophosphate-   BOP Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium    hexafluorophosphate-   RP Reversed Phase-   RT/rt room temperature-   SPPS Solid Phase Peptide Synthesis-   TBME tert-Butyl-methylether-   TFA trifluoroacetic acidTEMPO 2,2,6,6-Tetramethyl-1-piperidinyloxy,    free radical.-   RBF Round bottomed flask

For amino acid abbreviations see the table above.

-   Compounds Names . . .

The names of the open chain oligopeptides have been derived according torecommendations of the Joint Commission on Biochemical Nomenclature(“International Union of Pure and Applied Chemistry” and “InternationalUnion of Biochemistry”) published in Pure & Appl. Chem. 1984, 56,595-624. Previously, a simple peptide naming convention was used forthese compounds. The previous names are given in parenthesis.

Example 1 Synthesis of Compound A

1A Synthesis of Synthon 1

(i) Alternative 1:

1A(1) Preparation of Compound 1

-   (S)-Benzyl 2-((tert-butoxycarbonyl)amino)-5-hydroxypentanoate

Compound 1 was prepared by a procedure similar to the procedure reportedin R. K. Olson, K. Ramasamy, T. Emery, J. Org. Chem. 1984, 49, 3527.BOC-Glu-OBzl (50 g, 148.2 mmol) was dissolved in tetrahydrofuran (800mL) and triethylamine (47.3 g, 467.4 mmol) was added. The solution wascooled down to IT=−10° C. Ethyl-chloroformate (51.8 g, 98% purity, 467.8mmol) was added slowly, maintaining the temperature at IT=−10 to −15° C.The suspension thus obtained was stirred for an additional hour. IPC(HPLC) indicated the disappearance of the starting material. Thereaction mixture was allowed to warm-up to 0° C. and water (800 mL) wasadded at 0-5° C. within 25-30 minutes. Separation of the mixture into2-phases became visible. Under intensive stirring, sodium-borohydride(11.8 g, 299.4 mmol) was added in 10 portions at 0-5° C. Caution wasexercised as hydrogen gas evolved during addition of sodium-borohydride.The reaction mixture was stirred for additional 5 minutes at 0-5° C. andthe temperature was increased to 20-25° C. within 30 minutes. Cautionwas required as hydrogen-gas continued to evolve during temperatureincrease. The reaction mixture was stirred for 15 minutes at 20-25° C.before work-up.

For work-up, water (1250 mL) was added to the reaction mixture, followedby the addition of ethyl acetate (1250 mL). The phases were separatedand the aqueous phase was extracted with ethyl acetate (600 mL). Theorganic layers were combined and were washed with brine (2×600 mL). Theorganic layer was dried over magnesium sulfate, the solvent wasevaporated at reduced pressure and 40-45° C. to obtain 50.8 g crudeproduct. HPLC purity: 94 a %. The crude product was purified by flashchromatography on silica gel with hexane fraction/ethyl acetate (7:3 to1:1) as mobile phase.

Yield: 40.85 g (85.2%). Purity: 98% (HPLC). MS and NMR confirmed theproposed structure.

1A(2) Preparation of Compound 2: (S)-Benzyl2-((tert-butoxycarbonyl)amino)5-((tert-butyldiphenylsilyl)oxy)pentanoate

The alcohol (compound 1) from the previous step (40.3 g, 124.6 mmol) wasdissolved in dimethyl formamide (200 mL) and imidazole (12.8 g, 99.5%purity, 186.9 mmol) was added. The mixture was stirred at roomtemperature, until a solution was formed (5-10 min).tert.-Butyl-diphenyl-silyl-chloride (41.9 g, 98% purity, 149.5 mmol) wasadded dropwise within 10 minutes and stirring was continued foradditional 15 minutes at room temperature. IPC(HPLC) indicateddisappearance of the starting material (alcohol). For work-up, isopropylacetate (400 mL) was added to the reaction mixture, followed by the slowaddition of a half-saturated aq. sodium bicarbonate solution (400 mL).Caution was exercised as the addition was exothermic, gas evolutiontakes place. The phases were separated and the aqueous phase wasextracted with isopropyl acetate (400 mL). The organic layers werecombined and extracted with water (400 mL). The organic layer was driedon magnesium sulfate and the solvent was evaporated at 40-45° C. underreduced pressure. The residue was dried over night in vacuo at 25° C. toobtain 83.25 g crude product as colorless oil. HPLC analysis indicatedthe presence of 81 a % desired product and 18.6 a % of the correspondingsilanole. The crude product was used without further purification forthe next step. NMR and HR-MS of a purified sample confirmed the proposedstructure. HR-MS: calculated for C₃₃H₄₃NO₅Si: [M+H]⁺: 562.29833;[M+NH₄]⁺: 579.32488; [M+Na]⁺: 584.28027. Found: [M+H]⁺: 562.29848;[M+NH₄]⁺: 579.32489; [M+Na]⁺: 584.27995.

1A(3) Preparation of Compound 4

-   (S)-Benzyl    2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((tert-butyldiphenylsilyl)oxy)pentanoate

83.25 g crude product from the previous step (corresponding to 70 gtheoretical yield of compound 2, 124.6 mmol) was dissolved indichloromethane (650 mL) and trifluoroacetic acid (358.8 g, 99% purity,3115 mmol) was added dropwise to the intensively stirred solution. IPC(HPLC) after 30 minutes indicated complete cleavage of the BOCprotecting group. The reaction mixture (clear solution) was transferredinto a 6 L 4-bottomed flask with mechanical stirrer and was diluted with1000 mL of dichloromethane. Aqueous half saturated sodium carbonatesolution (1800 mL) was slowly added to the intensively stirred solution.Caution was exercised as strong gas evolution was observed duringaddition of sodium carbonate. pH of the aqueous phase after completionof the addition: 9-10. The reaction mixture was stirred for additional15 minutes and the phases were separated. The aqueous phase wasextracted with dichloromethane (1000 mL) and the organic layers werecombined to obtain a solution of the product in dichloromethane.

The solution was concentrated at 40-45° C. under reduced pressure to afinal volume of ca. 650 mL. for the next step. HPLC indicated thepresence of 80.5% of compound 3 and 19.5 a % silanole in the solution.

For FMOCylation, saturated aq. sodium bicarbonate solution (650 mL) wasslowly added to the intensively stirred solution of compound 3, followedby the addition of FMOC-chloride (36.55 g, 97% purity, 137 mmol).Caution was exercised as gas evolution was observed during the additionof FMOC-Cl. Stirring was continued for 15 minutes at room temperature.An IPC(HPLC) of the organic layer indicated disappearance of theintermediate compound 3 and complete conversion into compound 4. Forwork-up, the layers were separated and the aqueous phase was extractedwith dichloromethane (650 mL). The organic layers were combined and wereextracted with water (650 mL). The organic layer was dried overmagnesium sulfate and the solvent was removed at 40-45° C. under reducedpressure to obtain 112.6 g crude product.

The crude product was suspended in ethanol/isopropanol/water (89:5:6;2400 mL) and the suspension was heated to IT 50° C. to obtain asolution. The solution was cooled down to 45° C., seed crystals wereadded and the temperature was continued to cool down to 20-25° C. within1 hour. Crystallization started at ca. 40° C. The suspension was stirredat 20-25° C. over night, then cooled down to IT 0-5° C. within 30minutes and stirring was continued for additional 2 hours at 0-5° C. Theproduct was isolated by filtration, the filter cake was washed withethanol/isopropanol/water (89:5:6; 240 mL) and dried under reducedpressure to obtain pure compound 4 (100 a % purity according to HPLC).Yield: 66 g (77.4%). The product was fully characterized by MS and NMRHR-MS: Calculated for C₄₃H₄₅NO₅Si: [M+H]⁺: 684.31398; [M+NaH₄]⁺:701.34053; [M+Na]⁺: 706.29592. Found: [M+H]⁺: 684.31392; [M+NH₄]⁺:701.34021; [M+Na]⁺: 706.29539. The mother liquor gave 30.6 g of a foamcomprising 30 a % product and 29 a % silanole according to HPLC.

1A(4) Preparation of Synthon 1 from Compound 4

-   (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((tert-butyldiphenylsilyl)oxy)pentanoic    acid

Compound 4 (66 g, 96.6 mmol) was suspended inethanol/isopropylalcohol/water (89:5:6; 3000 mL) and the suspension washeated to IT 45° C. to obtain a solution. The solution was cooled downto IT 30° C. After inertization with Argon, palladium-catalyst (10% onbarium sulfate; 6.6 g) was added to the solution under an argon stream.The product was then hydrogenated under a hydrogen pressure slightlyabove the atmospheric pressure at 30-35° C. The hydrogenation wascompleted after 1.5 h according to HPLC. The reaction mixture wasfiltered over a cellulose based filter aid (Cellflock 40; cellulosebased filtering aid) and the filter aid was washed withethanol/isopropanol/water (89:5:6; 600 mL). Evaporation of the solventunder reduced pressure at 45-50° C. gave 59.58 g foam as crude product.The crude product was purified by chromatography on silica gel in 2portions (2×1 kg silica gel 60) using dichloromethane/methanol 95:5 to80:20 as mobile phase. Yield: 52 g (90.6%). The product was fullycharacterized by MS and NMR. HR-MS: Calculated for C₃₆H₃₉NO₅Si [M+H]⁺:594.26703; [M+NH₄]⁺: 611.29358; [M+Na]⁺: 616.24897. Found: [M+H]⁺:594.26743; [M+NH₄]⁺: 611.29385; [M+Na]⁺: 616.24900.

(ii) Alternative 2

1A(5) One-Pot Preparation of Compound 4 from Fmoc-Glu-OBzl Via Compound5

Fmoc-Glu-OBzl (5 g, 10.88 mmol) was dissolved in tetrahydrofurane (80mL) and triethylamine (3.3 g, 32.6 mmol) was added. The solution wascooled down to −15° C. and ethyl chloroformate (7.3 g, 67.27 mmol) wasadded to the solution over 30 minutes at −12° C. The suspension thusobtained was stirred for an additional hour at −10 to −15° C. and thetemperature was elevated to 0° C. Water (80 mL) was added dropwise tothe reaction mixture, maintaining the temperature at 0° C. Sodiumborohydride (total 0.805 g, 21.27 mmol) was added in 3 portions (1portion every 10 minutes) at 0° C. and the reaction mixture was stirredfor additional 1 h at 0° C. Caution was exercised as hydrogen gasevolved. The reaction mixture was diluted with water (100 mL) andextracted with isopropyl acetate (150 mL) and the layers were separated.The aqueous phase was extracted again with isopropyl acetate (100 mL)and the organic layers were combined. The combined organic phase waswashed with half saturated sodium chloride solution (2×50 mL) and thesolution was concentrated at reduced pressure to a final volume of ca.50 mL. The concentrated solution was clear-filtered and the filterresidue was washed with isopropyl acetate (20 mL). The solution ofcompound 5 thus obtained was transferred into a round bottomed flask andimidazole (1.49 g, 21.89 mmol) was added. The mixture was stirred for 15min at Rt and tert.-butyl-diphenyl-silyl-chloride (4.45 g, 16.19 mmol)was added. The reaction mixture was stirred for 15 h at rt. For work-up,the suspension was diluted with isopropyl acetate (20 mL) and extractedwith water (3×50 mL). The organic layer was separated and the solventwas evaporated under reduced pressure to obtain 9.95 g crude product.The crude product was purified by column chromatography on silicagelwith isopropyl acetate/hexane (2:8) as mobile phase to obtain 6.5 g of asolid, which was suspended in hexane and stirred for 3 h at rt. Theprecipitate was isolated by filtration and dried at 50° C. under reducedpressure to obtain 5.6 g of compound 4. Yield: 75% over two steps.HR-MS: calculated for C₄₃H₄₅NO₅Si: [M+H]⁺: 684.31398; [M+NH₄]⁺:701.34053; [M+Na]⁺: 706.29592. Found: [M+H]⁺: 684.31430; [M+NH₄]⁺:701.34073; [M+Na]⁺: 706.29577.

1A(6) Alternative One-Pot Preparation of Compound 4 from Fmoc-Glu-OBzlVia Compound 5 Using Isopropyl-Chloroformate Instead ofEthyl-Chloroformate

Fmoc-Glu-OBzl (60 g, 130.579 mmol) was dissolved in tetrahydrofuran (550mL) and triethylamine (40.8 g, 403.202 mmol) was added. A cloudysolution was obtained with some precipitate. This cloudysolution/suspension was transferred into a dropping funnel and was addedto a pre-cooled solution of isobutyl-chloroformate (54.96 g, 402.41mmol) in tetrahydrofuran (300 mL) in a 4.5 L reactor at −35 to −30° C.,maintaining this temperature during the addition. Residuals in thedropping funnel were washed with additional tetrahydrofuran (50 mL) andthe reaction mixture was stirred at −35 to −30° C. for another 2 hours.Water (960 mL) was added to the reaction mixture within 45 minutes,allowing the temperature to increase until 0° C. A suspension wasformed. Sodium borohydride (14.4 g, 380.625 mmol) was added in 20portions within 1 h at 0° C. and the reaction mixture was stirred for anadditional hour at 0° C. Caution was exercised as hydrogen gas evolved.

The suspension was poured onto t-butyl-methylether (600 mL) and thereaction flask was washed with water (600 mL), which was added to theproduct mixture (2-phases). The phases were separated, the water phasewas extracted with t-butyl-methylether (600 mL) and the organic phaseswere combined. The organic phase was washed with water (2×600 mL), driedover anhydrous magnesium sulfate (200 g) and the solvent was removedunder reduced pressure until a final volume of 1 L was achieved. Thesolution was diluted with dimethyl-formamide (600 g) and the solvent wasevaporated under reduced pressure, until a final volume of 400 mL isachieved. The solution of compound 5 thus obtained was transferred intoa round bottomed flask. Imidazole (14.4 g, 211.524 mmol) was added tothe DMF solution of compound 5 and the mixture was stirred for 5 minutesat rt. Finally, TBDPS-Cl (39.6 g, 144.07 mmol) is added dropwise during20 minutes at 20-25° C. and the reaction mixture is stirred for anadditional hour at this temperature.

The reaction mixture was then poured onto ethyl acetate (1200 mL) andthe mixture was extracted with water (700 mL). The layers were separatedand the organic layer was washed with water (3×300 mL). Evaporation ofthe solvent under reduced pressure gave 106 g crude product.

The crude product (106 g) was dissolved in Ethanol/Isopropanol/Water(89:5:6; 1200 mL) at 40-50° C. and seed crystals (0.5 g compound 4) wereadded. The mixture was allowed to cool down to room temperature andstirred for 17 hrs at rt. The suspension was cooled to −20° C. andstirred for 2 hrs at −20° C. The product was isolated by filtration, thefilter cake was washed with the solvent mixtureEthanol/Isopropanol/Water (89:5:6; 3×200 mL) and dried at 40° C. underreduced pressure to obtain 66.5 g of compound 4 (74.6% yield over 2steps). HPLC indicated >99 a % purity for the product.

Additional product can be isolated from the mother liquor (34 g afterevaporation of the solvent), which contains ca. 30 a % compound 4according to HPLC.

1B Synthesis of Precursor Peptide 1 by SPPS

Precursor Peptide 1: Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon1-H)-Leu-OH

Precursor Peptide 1 has been Prepared Using 2 Different Solid SupportsEquipment:

Solid phase synthesis reactor with a filter cloth or sintered glassfilter plate at the bottom. A nitrogen manifold allows to drain thereactor contents via filter cloth or sintered glass filter plate andbottom valve.

1B(1) Coupling of the Trityl-Linker to the Solid Support

200 g of Aminomethyl-polystyrene resin (crosslinked with 1% divinylbenzene, loading of aminomethyl groups 1 mmol/g) (supplier: SennChemicals AG, Dielsdorf/Switzerland) were stirred alternately withseveral portions of dimethylformamide (1600 mL) and isopropanol (1600mL) After two final washes with dimethlyformamide, the resin was treatedwith a previously prepared solution of 4-hydroxy-diphenylmethyl-benzoicacid (91.3 g 300 mmol), 1-hydroxy-benzotriazole monohydrate (45.9 g, 300mmol) and diisopropylcarbodiimide (75.7 g, 600 mmol) indimethylformamide (1600 mL). The reaction mixture was stirred for 1.5 hand a Ninhydrin test was performed. The test still showed free aminogroups and thus diisopropylcarbodiimide (7.6 g, 60 mmol) was added andthe reaction stirred over night. A further ninhydrin test in the morningwas negative and the reaction mixture was filtered off. The resin waswashed with dimethylformamide and isopropanol alternatingly. The resinwas dried in vacuo and yielded 257 g of dry linker-resin. The materialwas used for the next synthesis step without further analysis.

1B(2) Coupling of Fmoc-Leu-OH

Preparation of Fmoc-Leu-Linker-Resin

Linker-Resin (190 g, 147.8 mmol) was swollen by stirring in toluene(1400 mL). The solvent was filtered off and replaced by a solution oftoluene (1400 mL) and acetyl chloride (53 mL, 1478 mmol). This mixturewas stirred for 2 h, filtered off, replaced by an identical mixturewhich was stirred for another 2 h before filtering off. The chlorinatedresin was washed twice with toluene and three times withdichloromethane.

In a round bottom flask, a solution of Fmoc-Leu-OH (104.8 g, 296 mmol)and of N-Methylmorpholine (49 mL, 444 mmol) in dichloromethane (600 mL)was prepared. This solution was added to the resin and stirred overnight. In the morning, the solution was filtered of and the resin waswashed with dichloromethane and isopropanol alternatingly. The resin0was dried in vacuo and yielded 234.7 g of dry Fmoc-Leu-linker-Resin.The loading with Fmoc-groups was determined at 0.787 mmol/g what led toa yield of 185 mmol (125% of theory). Amino acid analysis at an externalcontractor confirmed <0.1% D-Leu enantiomer.

1B(3) Coupling of Fmoc-Thr-OH

Preparation of Fmoc-Thr-Leu-Linker-Resin

Fmoc-Leu-Linker-Resin (140 g, 109 mmol) was swollen by stirring in twosuccessive portions of dimethylformamide (1100 mL) for 30 min each.

Fmoc protecting group was cleaved by two subsequent washings of 20%piperidine in dimethylformamide for 5 min and 15 min respectively. Theresin was washed by several alternating washes with dimethylfomamide andisopropanol. Phenolphtalein and water were added to a sample of thefinal wash solution. The absence of pink colour confirmed successfulremoval of piperidine.

The resin was washed with tetrahydrofurane (1200 mL) three times toprepare for the following coupling step.

In a round bottomed flask a solution of Fmoc-Thr-OH (112.1 g, 328 mmol),hydroxybenzotriazole monohydrate (51.25 g, 334 mmol) anddiisopropylcarbodiimide (51 mL, 655 mmol) in tetrahydrofuran (600 mL)was prepared

The solution was added to the resin and the pH checked immediately(pH=6.5). The reaction mixture was stirred for 1.5 h until a ninhydrintest showed complete reaction. The solution was filtered off and theresin was washed with dimethylformamide and isopropanol alternatingly. Asmall sample of the resin was dried and sent for amino acid analysis(0.13% D-Leu, <0.1% D-Thr, <0.1% L-allo-Thr, <0.1% D-allo-Thr), the bulkof the material was subjected to the next step without further drying.

1B(4) Coupling of Fmoc-Gln(Trt)-OH

Preparation of Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin

The Fmoc-Thr-Leu-Linker-Resin from the previous step was swollen bystirring in two subsequent portions of dimethylformamide (1100 mL) for30 min each.

Fmoc protecting group was cleaved by two subsequent washings with 20%piperidine in dimethylformamide for 5 min and 15 min respectively. Theresin was washed by several alternating washes with dimethylfomamide andisopropanol. Phenolphtalein and water were added to a sample of thefinal wash solution. The absence of pink colour proved successfulremoval of piperidine.

The resin was washed with dimethylformamide (1100 mL) three times toprepare for the following coupling step.

In a round bottomed flask a solution of Fmoc-Gln(Trt)-OH (138.6 g, 226mmol), HATU (86.2 g, 226 mmol) and Ethyldiisopropylamine (58.4 g, 452mmol) in dimethylformamide (400 mL) was prepared.

The solution was added to the resin and the pH checked immediately(pH=10). The reaction mixture was stirred for 3 h until a ninhydrin testshowed complete reaction. The solution was filtered of and the resin waswashed with dimethylformamide and isopropanol alternatingly.

The resin was dried in vacuo and yielded 170 g of dryFmoc-Gln(Trt)-Thr-Leu-Linker-Resin. The loading with Fmoc-groups wasdetermined at 0.60 mmol/g indicating a yield of 102 mmol (94% of theoryover the last two steps). Amino acid analysis at an external contractorled to the following values: (0.13% D-Leu, <0.1% D-Thr, <0.1%L-allo-Thr, <0.1% D-allo-Thr, <0.8% D-Gln).

1B(5) Coupling of Isobutyric Acid

Preparation of Isobutyryl-Gln(Trt)-Thr-Leu-Linker-Resin

Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin (169 g, 101 mmol) was swollen bystirring in two subsequent portions of dimethylformamide (1300 mL) for30 min each.

Fmoc protecting group was cleaved by two subsequent washings with 20%piperidine in dimethylformamide (1300 mL) for 5 min and 15 minrespectively. The resin was washed by several alternating washes withdimethylfomamide and isopropanol. Phenolphtalein and water were added toa sample of the final wash solution. The absence of pink colour provedsuccessful removal of piperidine.

The resin was washed with dimethylformamide (1100 mL) three times toprepare for the following coupling step.

In a round bottom flask a solution of Isobutyric acid (17.9 g, 203mmol), PyBOP (105.5 g, 203 mmol) and Ethyldiisopropylamine (52.4 g, 406mmol) in dimethylformamide (550 mL) was prepared.

The solution was added to the resin and the pH checked immediately(pH=9.5). The reaction mixture was stirred for 2.5 h until a ninhydrintest showed complete reaction. The solution was filtered off and theresin was washed with dimethylformamide and isopropanol alternatingly.

The batch was directly subjected to the next step without drying andfurther analysis.

1B(6) Coupling of Fmoc-Ile-OH (Esterification)

Preparation of Isobutyryl-Gln(Trt)-Thr(Ile-Fmoc)-Leu-Linker-Resin

Isobutyryl-Gln(Trt)-Thr-Leu-Linker-Resin (wet from step above, 101 mmol)was swollen by stirring in three subsequent portions of dichloromethane(1200 mL) for 20 min each. The solvent was filtered off and MSNT (88 g,297 mmol) and Fmoc-Ile-OH (105 g, 297 mmol) were added as solids.Dichloromethane (500 mL) was added as well as a solution of N-methylimidazole (18.2 g, 223 mmol) and ethyldiisopropyamine (51.2 g, 396 mmol)in dichloromethane (100 mL) The reaction mixture was stirred for 2 huntil HPLC in process control showed complete reaction. The solution wasfiltered off and the resin was washed with three portions ofdichloromethane, three portions of dimethylformamide and three portionsof isopropanol subsequently. The resin was dried in vacuo and yielded172 g of dry Isobutyryl-Gln(Trt)-Thr(Ile-Fmoc)-Leu-Linker-Resin. Fmocloading was determined to be 0.418 mmol/g thus indicating a yield of 72mmol (71% over the last two steps).

1B(7) Coupling of Fmoc-N-methyl-Tyr(tBu)-OH

Preparation ofIsobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Fmoc)-Leu-Linker-Resin(Previously Named:Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Fmoc)-Leu-Linker-Resin)

Isobutyryl-Gln(Trt)-Thr(Ile-Fmoc)-Leu-Linker-Resin (172 g, 72 mmol) wasswollen by stirring in two subsequent portions of dimethylformamide(1300 mL) for 30 min each. Fmoc protecting group was cleaved by twosubsequent washings with 20% piperidine in dimethylformamide (1400 mL)for 5 min and 15 min respectively. The resin was washed by severalalternating washes with dimethylfomamide and isopropanol. Phenolphtaleinand water were added to a sample of the final wash solution. The absenceof pink colour proofed successful removal of piperidine.

The resin was washed with dimethylformamide (1100 mL) three times toprepare for the following coupling step.

A solution of Fmoc-N-methyl-Tyr(tBu)-OH (68.7 g, 144 mmol) and HATU(55.1 g, 144 mmol) in dimethylformamide (700 mL) was prepared and addedto the peptide-resin, followed by the addition of a solution ofEthyl-diisopropylamine (37.5 g 289 mmol) in dimethylformamide (100 mL)under stirring. pH checks immediately after addition of the couplingsolution and after 1 h of reaction gave the same result (pH 10) Thesolution was stirred for 2 h until a ninhydrin test showed completereaction. The solution was filtered of and the resin was washed withdimethylformamide and isopropanol alternatingly.

The batch was directly subjected to the next step without drying andfurther analysis.

1B(8) Coupling of Fmoc-Ile-OH

Preparation ofIsobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-IleFmoc)-Leu-Linker-Resin.(Previously Named:Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Ile-Fmoc)-Leu-Linker-Resin.)

The wet Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Fmoc)-Leu-Linker-Resin(72 mmol) was swollen by stirring in two subsequent portions ofdimethylformamide (1200 mL and 1300 mL) for 30 min each. Fmoc protectinggroup was cleaved by two subsequent washings with 20% piperidine indimethylformamide (1400 mL) for 5 min and 15 min respectively. The resinwas washed by several alternating washes with dimethylfomamide andisopropanol. Phenolphtalein and water were added to a sample of thefinal wash solution. The absence of pink colour proved successfulremoval of piperidine.

The resin was washed with dimethylformamide (1100 mL) three times toprepare for the following coupling step.

In a round bottom flask a solution of Fmoc-Ile-OH (103.9 g, 294 mmol)COMU (125.9 g, 294 mmol) and Ethyldiisopropylamine (76 g, 588 mmol) indichloromethane (440 mL) and dimethylformamide (440 mL) was prepared.

The solution was added to the resin and the reaction mixture stirred for20 h. After that time a ninhydrin test was done, showing completereaction. The solution was filtered off and the resin was washed withdimethylformamide and isopropanol alternatingly.

The resin was dried in vacuo and yielded 185.5 g of dryIsobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Fmoc)-Leu-Linker-Resin. Theloading with Fmoc groups was determined to be 0.40 mmol/g. Thus aquantitative yield of 74 mmol resulted.

1B(9) Coupling of Synthon 1 and Cleavage of Final Fmoc Protecting Group

Preparation of Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon1-H)-Leu-Linker-Resin

(Previously Named: Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr-Ile-Synthon1-H)-Leu-Linker-Resin)

Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Fmoc)-Leu-Linker-Resin (30 g,12 mmol) was swollen by stirring in two subsequent portions ofdimethylformamide (240 mL and 250 mL) for 30 min each.

Fmoc protecting group was cleaved by two subsequent washings with 20%piperidine in dimethylformamide (250 mL) for 5 min and 15 minrespectively. The resin was washed by several alternating washes withdimethylfomamide and isopropanol. Phenolphtalein and water were added toa sample of the final wash solution. The absence of pink colour provedsuccessful removal of piperidine.

The resin was washed with dimethylformamide (250 mL) three times toprepare for the following coupling step.

In a round bottom flask, a solution of synthon 1 (14.6 g, 24.6 mmol)PyBOP (12.85 g, 24.6 mmol) and Ethyldiisopropylamine (6.4 g, 49.2 mmol)in dimethylformamide (120 mL) was prepared.

The solution was added to the resin and the reaction mixture stirred for3 h. After that time a ninhydrin test confirmed complete reaction. Thesolution was filtered off and the resin was washed withdimethylformamide and isopropanol alternatingly.

The resulting peptide-resin was swollen by stirring in two subsequentportions of dimethylformamide (250 mL) for 30 min each.

Fmoc protecting group was cleaved by two subsequent washings with 20%piperidine in dimethylformamide (250 mL) for 5 min and 15 minrespectively. The peptide-resin was washed by several alternating washeswith dimethylfomamide and isopropanol. Finally the peptide-resin waswashed three times with dichloromethane (250 mL) to prepare for thecleavage of the peptide.

1B(10) Cleavage of Precursor Peptide 1 from Solid Support

Preparation of Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon1-H)-Leu-OH (=Precursor Peptide 1)

(Previously Named: Isobutyryl-Gln(Trt)-Thr(Ile-N-Me-Tyr(tBu)-Ile-Synthon1-H)-Leu-OH)

To the wet Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon1-H)-Leu-Linker-Resin, a mixture of acetic acid (125 mL) anddichloromethane (125 mL) was added and the suspension stirred for 2 h.The suspension was filtered and the filtrate collected in a roundbottomed flask (filtrate 1). The resin was washed twice withdichloromethane (250 mL) and the washes were combined with filtrate 1.

The resin was treated with a fresh portion of acetic acid (125 mL) anddichloromethane (125 mL) for another 2 h. The suspension was filteredand the filtrate collected in a round bottomed flask (filtrate 2). Theresin was washed twice with dichloromethane (250 mL) and the washescombined with filtrate 2.

Acetic acid (200 mL) and dichloromethane (50 mL) were added to the resinand the suspension stirred over night. The suspension was filtered andthe filtrate collected as filtrate 3.

The three filtrates were worked up separately to assess theeffectiveness of the cleavage method. The filtrates were concentrated ina rotary evaporator and the residual acetic acid removed by azeotropicdistillation with three portions of toluene (100 mL). The oily residueswere then dried in high vacuo at a lyophilisator. Yield: filtrate 1:12.5 g, filtrate 2: 2.1 g, filtrate 3: 0.3 g.

The crude material, Precursor peptide 1, was purified byRP-chromatography, the fractions concentrated in a rotary evaporator andthe concentrate freeze dried. Purity: 98.8% Yield: 8.5 g (48% for thelast two steps). The product was characterized by, ¹H-NMR, ¹³C-NMR andHR-MS. The spectra confirmed the proposed structure. The NMR spectraindicated the presence of several conformations.

HR-MS: calculated for C₈₅H₁₁₆N₈O₁₃Si: [M+H]⁺: 1485.85039. Found: [M+H]⁺:1485.84929.

Second SPPS-Synthesis of Precursor Peptide 1 Using a Chlorinated Resin

1B(11) Immobilization of the First Amino Acid (Fmoc-Leu-OH)

2-Chlorotrityl chloride resin (30 g, L=1.2 mmol/g; Merck Novabiochem855017) was swelled with 240 ml of dried DCM for 10 min and then thesolvent was drained, the solid phase reactor was kept close under a softnitrogen stream. In the meantime, in a round bottom flask were mixedFmoc-Leu-OH (42.6 g, 120.8 mmol) and 100 ml of dry 1,4-Dioxane. Thesolvent was evaporated under vacuum at 45° C., then a second portion of1,4 dioxane was added. The solvent was evaporated again under vacuum at45° C. until a colorless oil residue was obtained. To this oily residuewere then added sequentially dry DCM (155 mL) and DIPEA (40.4 mL), thesolution was added in one single portion to the pre-swollen resin andthen 5 min later a second portion of DIPEA (16.5 mL) was added. Thesuspension was stirred for 2 h and the reaction medium was drained. Inorder to quench potential non-reacted chloride, 240 ml of a quenchingsolution of DCM/MeOH/DIPEA (70/15/15, v/v) was added and the mixture wasstirred for 10 min. A loading of 0.95 mmol/g was measured.

General Protocol for Fmoc Cleavage:

To the pre-swollen resin were added 240 ml of a solution of 25%piperidine in DMF, the suspension was stirred for 5 minutes and then thereaction mixture was removed by filtration, then a second portion of thesame solution of piperidine was added, the suspension was stirred for 15minutes and then the solvent was removed by filtration.

Resin washing with drying down:

The resin was washed as follows:

-   250 ml of DMF (6×2 min)-   250 ml of IPA (3×2 min)-   250 ml of TBME (6×2 min)

The resin was dried overnight under vacuum at 40 C.° to obtain 35.7 g ofH-Leu-Resin.

1B(12): SPPS Synthesis of H-Thr-Leu-Resin H-Leu-Resin (30 g) from theprevious step was swollen with 270 ml of DMF for 30 min then the solventwas drained.

In a round bottomed flask (RBF) were mixed Fmoc-Thr-OH (25.0 g, 73.2mmol), BOP (40.4 g, 91.5 mmol) and DMF (250 mL), the mixture was stirredfor 2 min and then DIPEA (18.9 g, 146.4 mmol) was added. The mixturethus obtained was added in one portion to the pre-swollen peptide-resinand the reaction mixture was stirred for 2.5 h. The Kaiser test waspositive and therefore a second coupling was done according to theconditions previously described. After 1.0 h the Kaiser test wasnegative and the reaction was considered to be complete and the reactionmedium was removed via filtration.

The resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (5×2 min).

The resin was dried over night under vacuum at 40 C.° yielding 52.2 g ofFmoc-Thr-Leu-Resin ready for the Fmoc-cleaving step.

Fmoc Cleavage:

The resin (52.2 g) was suspended in 272 ml of DMF and stirred for 1.0 hthen the solvent was removed by filtration.

The Fmoc protecting group was cleaved in the same manner as describedfor 1B(11) and the peptide-resin was washed as follows:

-   250 ml DMF (6×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (6×2 min)

The resin was dried overnight under vacuum at 40 C.° yielding 39.9 g ofH-Thr-Leu-Resin

1B(13): SPPS Synthesis of H-Gln(Trt)-Thr-Leu-Resin

H-Thr-Leu-Resin from the previous step (39.9 g) was swollen with 270 mlof DMF for 30 min then the solvent was drained.

In an RBF were mixed Fmoc-Gln(Trt)-OH (44.7 g, 73.2 mmol), BOP (40.4 g,91.5 mmol) and 250 ml of DMF. The mixture was stirred for 2 min and thenDIPEA (18.9 g, 146.4 mmol) was added. The resulting mixture was added inone portion to the pre-swollen H-Thr-Leu-Resin and the reaction mixturewas stirred for 2.5 h, the Kaiser test was negative, then the reactionwas considered to be complete and the reaction medium was removed viafiltration.

The resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (3×2 min)-   250 ml DMF (4×2 min)

The wet peptide-resin was used for the Fmoc-cleaving step without anyfurther manipulation.

Fmoc Cleavage:

The fmoc protecting group was cleaved according to the proceduredescribed in step 1B(11). Then the resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (5×2 min)

The resin was dried overnight under vacuum at 40 C.° yielding 54.4 g ofH-Gln(Trt)-Thr-Leu-Resin.

1B(14): SPPS Synthesis of Isobutyryl-Gln(Trt)-Thr-Leu-Resin

The peptide-resin from the previous step (54.4 g) was swollen with 270ml of DMF for 30 min, then the solvent was drained.

In a RBF were mixed Isobutyric acid (6.4 g, 73.2 mmol), PyBop (38.1 g,73.2 mmol) and 230 ml of DMF, the mixture was stirred for 2 min and thenDI PEA (28.3 g, 219.6 mmol) was added. The solution was added in oneportion to the pre-swollen peptide-resin and the reaction mixture wasstirred for 1.5 h. The Kaiser test was negative, then the reaction wasconsidered to be complete and the reaction medium was removed viafiltration.

The resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (5×2 min).

The resin was dried overnight under vacuum at 40 C.° yielding 55.8 g ofIsobutyryl-Gln(Trt)-Thr-Leu-Resin.

1B(15): SPPS Synthesis of Isobutyryl-Gln(Trt)-Thr(Ile-H)-Leu-Resin

The peptide-resin coming from the previous step (55.8 g) was suspendedin 250 ml of dry DCM for 1.0 h. Then the solvent was removed byfiltration and the wet peptide-resin was kept under a soft stream ofnitrogen and the temperature was adjusted to 0-5° C.

In a RBF were mixed Fmoc-Ile-OH (51.7 g, 146.4 mmol), and 150 ml of drydioxane. The solvent was evaporated under vacuum at 45° C. until an oilyresidue was observed. The distillation process was repeated and then tothe residue were added 150 ml of dried DCM, the solution was cooled to−10° C. and MSNT (43.3 g, 146.4 mmol) was added, the suspension wasstirred for 3 min and then N-methyl-imidazole (14.2 g, 173 mmol) wasadded, the mixture was stirred for 2 min and the solution was addeddropwise in 10 min to the above prepared peptide-resin. After theaddition was finished the suspension was stirred under a nitrogenatmosphere for 2.0 h

The resin was washed as follows:

-   250 ml DMC (4×2 min)-   250 ml DMF (3×2 min)

The wet peptide-resin was used for the Fmoc-cleaving step without anyfurther manipulation.

Fmoc Cleavage:

The Fmoc protecting group was cleaved according to the general protocoldescribed in 1B(11)

After the Fmoc cleavage was done, the resin was washed as follows:

-   250 ml DMF (5×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (5×2 min)

The resin was dried overnight under vacuum at 40 C.° yielding 57.2 g ofIsobutyryl-Gln(Trt)-Thr(O-Ile-H)-Leu-Resin.

1B(16): SPPS Synthesis ofIsobutyryl-Gln(Trt)-Thr(O-Ile-Tyr(tBu)MeN)-Leu-Resin

The peptide-resin coming from the previous step (57.2 g) was suspendedin 250 ml DMF for 30 min. for swelling, then the solvent was removed byfiltration.

In a RBF were mixed Fmoc-NMeTyr(tBu)-OH (52.0 g, 109.8 mmol), HATU (41.7g, 109.8 mmol) and 230 ml of DMF and the mixture was stirred for 2 min.Then DI PEA (28.3 g, 219.6 mmol) was added and the solution was stirredfor 2.0 min. The resulting solution was then added to the pre-swollenpeptide-resin.

The reaction mixture was stirred for 1.0 h and a Kaiser-test wasperformed. The Kaiser test was negative. The reaction was considered tobe complete and the reaction medium was removed via filtration.

The peptide-resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (1×2 min)

The wet peptide-resin was used for the Fmoc-cleaving step without anyfurther manipulation.

Fmoc Cleavage:

The Fmoc protecting group was cleaved according to the general protocolused in 1B(11).

After the Fmoc cleavage was done, the resin was washed as follows:

-   250 ml DMF (5×2 min)-   250 ml IPA (1×2 min)-   250 ml DMF (5×2 min)

The obtained isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-H)-Leu-Resin wasused for the next step without drying.

1B(17): SPPS Synthesis ofIsobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-H)-Leu-Resin

Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-H)-Leu-Resin from the previousstep was swollen with 270 ml of DMF for 30 min, then the solvent wasdrained.

In a round bottomed flask were mixed Fmoc-Ile-OH (38.8 g, 109.8 mmol),HATU (41.7 g, 109.8 mmol) and DMF (250 mL), the mixture was stirred for2 min and then DI PEA (28.3 g, 219.6 mmol) was added. The solution wasadded in one portion to the pre-swollen peptide-resin and the reactionmixture was stirred for 2.0 h, The Chloranil test was positive and thena second coupling with Fmoc-Ile-OH was performed. 3 h after the secondcoupling, the peptide-resin was isolated by filtration.

The peptide-resin was washed as follows:

-   250 ml DMF (4×2 min)-   250 ml IPA (3×2 min)-   250 ml DMF (4×2 min)

The wet peptide-resin was used for the Fmoc-cleaving step without anyfurther manipulation.

Fmoc Cleavage:

The Fmoc protecting group was removed in the same manner as described in1B(11), and the peptide-resin was washed as follows.

-   250 ml DMF (5×2 min)-   250 ml IPA (3×2 min)-   250 ml TBME (4×2 min).

The peptide-resin was dried overnight under vacuum at 40 C.° yielding67.0 g of Isobutyryl-Gln(Trt)-Thr(I le-Tyr(tBu)Me-Ile-H)-Leu-Resin.

1B(18): SPPS Synthesis ofIsobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-Resin

Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-H)-Leu-Resin from theprevious step (33.5 g) was suspended in 150 ml of DMF for 30 min thenthe solvent was drained. In a RBF were mixed Fmoc-Synthon 1-OH (34.3 g,57.8 mmol), PyBop (30.0 g, 57.7 mmol) and 113 ml of DMF, the mixture wasstirred for 2 min and then DIPEA (14.9 g, 115.2 mmol) was added. Thesolution was added in one portion to the pre-swollen peptide-resin andthe reaction mixture was stirred for 2.0 h. The reaction medium wasremoved by filtration.

The peptide-resin was washed as follows:

-   150 ml DMF (4×2 min)-   150 ml IPA (3×2 min)-   150 ml DMF (4×2 min)

The wet peptide-resin was used for the Fmoc-cleaving step without anyfurther manipulation.

Fmoc Cleavage:

The Fmoc protecting group was removed in the same way as in 1B(11) butusing 150 ml of the piperidin solution, and the peptide-resin was washedas follows.

-   150 ml DMF (5×2 min)-   150 ml IPA (3×2 min)-   150 ml TBME (4×2 min)

The peptide-resin was dried overnight under vacuum at 40 C.° yielding36.0 g of Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon1-H)-Leu-Resin.

1B(19): Alternative SPPS Synthesis: Cleavage of Peptide from SolidSupport

Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-Resin fromthe previous step (29.75 g) was treated with 350 ml of dried DCM for 1.0h, the solvent was removed by filtration and then 350 ml of a solutionof 30% v/v HFIP in DCM was added. The mixture was stirred for 10 min andthen the solvent was removed by filtration and kept aside. To the wetresin was added a second portion of the same HFIP solution and thesolution was stirred for 10 min, then the solvent was removed byfiltration and pooled with the previous solution. The resin was washed 3times with DCM (350 ml) and the washes were combined with the cleavagesolutions.

The combined solution was concentrated under vacuum until an oilyresidue was observed and then 200 ml of toluene was added, the solventwas evaporated under reduced pressure at 45° C. until an oily residuewas obtained. 350 ml of hexane was added and the suspension was stirredfor 2 h. The solvent was removed by filtration and the filter cake waswashed with hexane (50 ml), the wet cake was dried overnight undervacuum at 35° C. to yield 19.24 g of crude precursor peptide 1(=Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-OH).

Part of the crude precursor peptide 1 (6.0 g) was purified byRP-chromatography, the product fractions were concentrated in a rotaryevaporator and the concentrate was freeze dried to obtain 2.2 g of pureprecursor peptide 1 in 99.3% a purity. Product containing side fractionswere treated similarly to obtain additional 1.25 g of less pureprecursor peptide in 71.4% a purity. Extrapolated for the whole amountof crude precursor peptide 1 (19.24 g), this would correspond to 7.05 gpure precursor peptide 1 from major fractions and additional 4.01 gprecursor peptide 1 (in 71.4% a purity) from side fractions.

1C Solution Phase Synthesis of Compound 8

1C(1) Synthesis of Compound 6

(S)—N¹-((3S,6S,9S,12S,15S,18S,19R)-6-(4-(tert-butoxy)benzyl)-3,9-di((S)-sec-butyl)-12-(3-((tert-butyldiphenylsilyl)oxy)propyl)-15-isobutyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-2-isobutyramido-N⁵-tritylpentanediamide

A cloudy solution/suspension of Precursor peptide 1 (1.9 g, 1.28 mmol)in acetonitrile (120 mL) was added during 90 min to a stirred mixture ofHATU (972 mg, 2.56 mmol) and DMAP (468.6 mg, 3.84 mmol) in acetonitrile(100 mL) at 35° C. The dropping funnel was washed with acetonitrile (30mL). An IPC(HPLC) after the addition of the suspension indicated theabsence of the precursor peptide and the completion of the cyclization.For work-up, the solvent was evaporated under reduced pressure until afinal volume of ca. 50 mL and the residue was diluted with isopropylacetate (250). The organic phase was extracted with water (2×100 mL) andthe solvent was evaporated under reduced pressure to obtain 2.3 g ofcrude product. The crude product was purified by flash-chromatography onsilicagel with ethyl acetate as mobile phase to obtain 1.79 g (1.22mmol) compound 6 as a foam. Yield: 95%. The product was characterizedby, ¹H-NMR ¹³C-NMR and HR-MS. The spectra confirmed the proposedstructure. NMR spectra indicated the presence of several conformations.

HR-MS: calculated for C₈₅H₁₁₄N₈O₁₂Si: [M+H]⁺:1467.83983;[M+NH₄]⁺:1484.86637; [M+Na]⁺: 1489.82177. Found: [M+H]⁺:1467.83984;[M+NH₄]⁺:1484.86555; [M+Na]⁺: 1489.82073.

Second Example for the Synthesis of Compound 6 (15 g Scale)

A solution of the precursor peptide 1 (15.0 g) in t-butyl-methyl-ether(750 mL) was added slowly during 1.5 h to a pre-cooled solution of DMAP(2.80 g) and HATU (5.87 g) in acetonitrile (375 mL) at 0° C. Thereaction mixture was stirred for additional 30 min at room temperature.The reaction mixture was then diluted with t-butyl-methyl-ether (750 mL)and poured onto half-saturated aq. NaCl-solution (1500 mL). The phaseswere separated and the organic phase was extracted again withhalf-saturated aq. NaCl-solution (1500 mL). The organic layer wasseparated and the solvent was partly evaporated under reduced pressureto a final volume of ca. 175 mL. This solution was filtered over silicagel (column with 225 g silica gel) using t-butyl-methyl-ether as mobilephase. Evaporation of the solvent and drying in vacuo at 40-45° C. gavecompound 6 in 97.53% purity according to HPLC. Yield: 14.19 g (95.7%).

1C(2) Synthesis of Compound 7

(S)—N¹-((3S,6S,9S,12S,15S,18S,19R)-6-(4-(tert-butoxy)benzyl)-3,9-di((S)-sec-butyl)-12-(3-hydroxypropyl)-15-isobutyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-2-isobutyramido-N⁵-tritylpentanediamide

Compound 6 (1.79 g, 1.22 mmol) was dissolved in tetrahydrofurane (60 mL)and Et₃N(HF)₃ (6.62 g, 41.1 mmol) was added at room temperature. Thereaction mixture was stirred at room temperature for 8.5 hours, thendiluted with isopropyl acetate (200 mL), and the resultingsolution/suspension was slowly added to an intensively stirred saturatedaq. NaHCO₃-solution. The organic phase was separated and extracted withwater (100 mL). Evaporation of the solvent under reduced pressure gave1.86 g crude product, which was purified by flash-chromatography onsilica gel with ethyl acetate/isopropanol (95:5) as mobile phase toobtain 1.24 g (1.00 mmol) of compound 7.

Yield: 82%.

The product was fully characterized by IR, NMR and MS. The spectraconfirmed the proposed structure.

HR-MS: calculated for C₆₉H₉₆N₈O₁₂: [M+H]⁺:1229.72205; [M+NH₄]⁺:1246.74860; [M+Na]⁺: 1251.70399. Found: [M+H]⁺: 1229.72128;[M+NH₄]⁺:1246.74780; [M+Na]⁺: 1251.70310.

Second Example for the Preparation of Compound 7 (14 g Scale)

Compound 6 (14.0 g, 9.537 mmol) was dissolved in tetrahydrofuran (220mL) and t-butyl-methyl-ether (116 mL) was added. The solution wastreated with Et₃N(HF)₃ (23.05 g) by slow addition within 10 minutes. Thereaction mixture was stirred for 24 h at room temperature. For work-upthe reaction mixture was diluted with t-butyl-methyl-ether (570 mL) andthe mixture was poured onto half saturated aq. NaHCO₃-solution (632 mL).The biphasic mixture was stirred for 30 min and the phases wereseparated. The organic phase was extracted with water (280 mL). Bothaqueous phases were extracted with t-butyl-methyl-ether (380 mL) and theorganic phases were combined. The organic phase was dried over anhydrousMgSO₄ (8.0 g) and the solvent was partly evaporated under reducedpressure to a final volume of ca. 100 mL. Toluene (140 mL) was added andthe solvent was evaporated again to a final volume of 80 mL. Thissolution was diluted with t-butyl-methyl-ether (70 mL) and the productwas precipitated by slow addition of heptanes (140 mL) during 30 min.The formed suspension was heated to 50-55° C. and stirred for 30 min atthis temperature. The suspension was then cooled down to 0° C. within 30min, stirred at 0° C. for 2 h and the product was isolated byfiltration. The product, a white precipitate, was dried in vacuo toobtain 11.08 g of compound 7. (94.5% yield). HPLC of the productindicated 97 a % purity.

1C(3) Synthesis of Compound 8 (See Reaction Scheme 4 for Structure)

(S)—N¹-((3S,6S,9S,12S,15S,18S,19R)-6-(4-(tert-butoxy)benzyl)-3,9-di((S)-sec-butyl)-15-isobutyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-12-(3-oxopropyl)-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-2-isobutyramido-N⁵-tritylpentanediamide

Compound 7 (1.2 g, 0.98 mmol) was dissolved in a mixture oftetrahydrofuran (190 mL) and dimethyl sulfoxide (62 mL).1-Hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (IBX) (2.42 g, 45% g/g, 3.9mmol) was added and the solution was stirred for ca. 4.5 hours, afterwhich time HPLC indicated disappearance of the starting material(compound 7). The reaction mixture was then poured onto saturated, aq.NaHCO₃-solution (300 mL) and was extracted with dichloromethane (2×300mL). The organic layers were combined and were washed with water (2×300mL). Evaporation of the solvent under reduced pressure gave 2.31 g ofcrude product as a foam. The crude product was purified byflash-chromatography on silicagel with ethyl acetate/isopropanol (95:5)to obtain 1.16 g of a product mixture, comprising at least 2 productswith the desired mass in LC-MS. The product mixture was used as such forthe next step.

HR-MS (major isomer): calculated for C₆₉H₉₄N₈O₁₂: [M+H]⁺: 1227.70640;[M+NH₄]⁺: 1244.73295; [M+Na]⁺: 1249.68834. Found: [M+H]⁺: 1227.70599;[M+NH₄]⁺: 1244.73200; [M+Na]⁺: 1249.68733.

HR-MS (minor isomer): calculated for C₆₉H₉₄N₈O₁₂: [M+H]⁺: 1227.70640;[M+NH₄]⁺: 1244.73295; [M+Na]⁺: 1249.68834. Found: [M+H]⁺: 1227.70598;[M+NH₄]⁺: 1244.73198; [M+Na]⁺: 1249.68751.

Second Example for the Synthesis of Compound 8 (10 g Scale)

Compound 7 (10.0 g, 8.13 mmol) was dissolved in tetrahydrofuran (127 mL)and dimethylsulfoxide (42 mL). To this solution,1-Hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (IBX) (15.18 g, 45% m/m,24.4 mmol) was added under intense stirring at room temperature. Thereaction mixture was stirred for 16 h at room temperature. The reactionmixture was then poured onto aq. NaHCO₃-solution (500 mL) and ethylacetate (250 mL) was added. The biphasic mixture was stirred for 30minutes and the layers were separated. The organic phase was washed withhalf-saturated aq. NaCl-solution (250 mL) and the layers were separated.The water layers were extracted with ethyl acetate ((250 mL) and theorganic phases were combined. The combined organic phase was dried onanhydrous magnesium sulfate and the solvent was partly evaporated toobtain ca. 50 g solution. This solution was flashed over a silica gelcolumn (100 g silica gel) using ethyl acetate/methanol (98:2 v/v) asmobile phase. The product solution (537.4 g) thus obtained was treatedwith toluene (135 mL) and the resulting solution was concentrated to 112g final weight by partly evaporation of the solvent at 45° C. underreduced pressure. The resulting solution was cooled to 0° C. andheptanes (135 mL) was added during 30 min. The suspension thus obtainedwas stirred for 1 h at 0° C., the product was isolated by filtration andwashed with heptanes (2×30 mL). Finally the product was dried in vacuoat 45° C. over night to obtain 8.824 g of a mixture of compound 8 andits hemiaminal-isomers. Yield: 88.4%.

1D Synthesis of Compound A

(S)—N¹-((2S,5S,8S,11R,12S,15S,18S,21R)-2,8-di-(S)-sec-butyl-21-hydroxy-5-(4-hydroxybenzyl)-15-isobutyl-4,11-dimethyl-3,6,9,13,16,22-hexaoxo-10-oxa-1,4,7,14,17-pentaazabicyclo[16.3.1]docosan-12-yl)-2-isobutyramidopentanediamide

Compound 8 (2.0 g) was dissolved in dichloromethane (400 mL) and thesolution was cooled down to 0° C. Trifluoroacetic acid (115.9 g) wasadded to the stirred solution at 0° C. and the reaction mixture wasstirred for 4 h at 0° C. Dichloromethane (400 mL) was added at thistemperature, followed by the addition of water (20 g). The reactionmixture was allowed to warm-up to room temperature and stirring wascontinued for additional 5 hours at room temperature. For work-up, thereaction mixture was poured onto a stirred solution of sodium acetate(165.1 g) in water (800 mL) and ethyl acetate (400 mL) was added toobtain a solution. The upper layer (aqueous phase) was removed and thelower organic phase (dichloromethane phase) was washed with water (2×200mL). The water layers were extracted with ethyl acetate (200 mL) and theorganic layers were combined. The solvent was removed under reducedpressure to obtain crude Compound A as a mixture of 5- and 6-ringisomers (see Reaction Scheme 5), accompanied by trityl alcohol and otherbyproducts of the reaction.

The crude product was purified by RP-chromatography on Silica Kromasil100-10-C8 with a gradient of acetonitrile/water as mobile phase. Theproduct containing fractions were evaporated to remove acetonitrile, theprecipitate was dissolved in ethyl acetate and the solvent was removedunder reduced pressure to obtain 0.895 g of compound A; yield: 59.1%.The product was fully characterized by NMR and MS and the spectra of theproduct were identical to Compound A from fermentation.

HR-MS: Calculated for C₄₆H₇₂O₁₂N₈: [M+H]⁺: 929.53425; [M+NH₄]⁺:946.56080; [M+Na]⁺: 951.51619. Found: [M+H]⁺: 929.53445; [M+NH₄]⁺:946.56129; [M+Na]⁺: 951.51624.

¹H-NMR (600 MHz, d₆-DMSO) δ_(H): −0.11 (3H, d, J=6.2 Hz), 0.64 (4H, m),0.77 (3H, d, J=6.2 Hz), 0.81 (3H, t, J=7.3 Hz), 0.84 (3H, d, J=7.0 Hz),0.88 (3H, d, J=6.6 Hz), 1.02 (3H, d, J=6.7 Hz), 1.02 (1H, m), 1.03 (3H,d, J=6.7 Hz), 1.09 (1H, m), 1.20 (3H, d, J=6.2 Hz), 1.24 (1H, m), 1.39(1H, m), 1.51 (1H, m), 1.75 (6H, m), 1.83 (1H, m), 1.92 (1H, m), 2.12(2H, m), 2.47 (1H, m), 2.58 (1H, m), 2.67 (1H, m), 2.71 (3H, s), 3.16(1H, d, J=14.2 Hz), 4.30 (1H, m), 4.34 (1H, m), 4.42 (1H, d, J=10.6 Hz),4.45 (1H, m), 4.61 (1H, d, J=9.2 Hz), 4.71 (1H, dd, J=9.5, 5.5 Hz), 4.93(1H, s), 5.05 (1H, dd, J=11.4, 2.6 Hz), 5.48 (1H, m), 6.07 (1H, d, J=2.6Hz), 6.64 (2H, d, J=8.4 Hz), 6.73 (1H, s), 6.99 (2H, d, J=8.4 Hz), 7.25(1H, s), 7.35 (1H, d, J=9.2 Hz), 7.64 (1H, d, J=9.5 Hz), 7.73 (1H, d,J=9.2 Hz), 8.01 (1H, d, J=7.7 Hz), 8.42 (1H, d, J=8.8 Hz), 9.17 (1H, s).

¹³C-NMR (150 MHz, d₆-DMSO) δ_(C): 10.35, CH₃; 11.21 CH₃; 13.85, CH₃;16.00, CH₃; 17.68, CH₃; 19.52, 2×CH₃; 20.89, CH₃; 21.75, CH₂; 23.30,CH₃; 23.74, CH₂; 24.21, CH; 24.48, CH₂; 27.35, CH₂; 29.78, CH₂; 30.08,CH₃; 31.49, CH₂; 33.18, CH; 33.24, CH₂; 33.76, CH, 37.41, CH; 39.23,CH₂; 48.84, CH; 50.69, CH; 52.11, CH; 54.17 CH; 54.70, CH; 55.31, CH;60.66, CH; 71.89, CH; 73.97, CH; 115.32, 2×CH; 127.34, Cq; 130.37, 2×CH;156.27, Cq; 169.12, Cq; 169.29, Cq; 169.37, Cq; 169.79 Cq; 170.65, Cq;172.40, Cq; 172.53, Cq; 173.87 Cq; 176.38, Cq.

Second Example for the Synthesis of Compound A (8.5 g Scale)

Compound 8 (8.5 g, 6.924 mmol) was dissolved in dichloromethane (595 mL)and the solution was cooled to 0° C. A solution of trifluoroacetic acid(127.5 mL) in dichloromethane (127.5 mL) was added to the cooledsolution, maintaining the temperature at 0-5° C. The reaction mixturewas stirred for 5.5 h at 0° C. and was diluted with dichloromethane (850mL), followed by addition of water (42.5 mL). The reaction mixture wasallowed to warm up to room temperature and was stirred for 17.5 h atroom temperature. For work-up, the reaction mixture was poured onto astirred, biphasic mixture of aqueous sodium acetate (169 g sodiumacetate in 722 g water) and ethyl acetate (700 mL). The phases wereseparated and the organic phase was washed again with aqueous sodiumacetate solution (169 g sodium acetate in 722 g water). The layers wereseparated and the organic phase was washed with water (2×850 mL). Theindividual aqueous phases were extracted with ethyl acetate (700 mL) andthe organic layers were combined. The organic layer was dried overanhydrous magnesium sulfate and the solvent was partly evaporated at40-45° C. under reduced pressure to obtain 187 g of a thin suspension.Heptane (187 g) was added within 15 minutes and the suspension formedwas cooled to 0° C. The suspension was stirred for 1 h at 0° C. and theproduct was isolated by filtration. The crude product was dried in vacuoat 40° C. to obtain 5.65 g crude compound A. Crude yield: 87.8%.

0.96 g of the crude product was purified by RP-chromatography on SilicaKromasil 100-10-C8 with a gradient of acetonitrile/water as mobilephase. The product containing fractions were evaporated to removeacetonitrile, the precipitate was dissolved in ethyl acetate and thesolvent was partly removed under reduced pressure to obtain 40 g of asolution. Heptane (40 g) was added to the stirred solution during 30 minat room temperature and the resulting suspension was stirred at roomtemperature for additional 2 h. The product was isolated by filtrationand washed with ethyl acetate/heptane (1:1, 2×10 mL). The product wasdried in vacuo at 40-45° C. for 16 h to obtain 0.702 g of compound A.Yield after RP-chromatography and precipitation: 64%.

Example 2 Synthesis of Compound B (Nostopeptin BN920)(S)-2-acetamido-N¹-((1S,2S,5S,8S,11R,12S,15S,18S,21R)-2-benzyl-21-hydroxy-5-(4-hydroxybenzyl)-15-isobutyl-8-isopropyl-4,11-dimethyl-3,6,9,13,16,22-hexaoxo-10-oxa-1,4,7,14,17-pentaazabicyclo[16.3.1]docosan-12-yl)pentanediamide

Compound B (Nostopeptin BN920)

2A Synthesis of Precursor Peptide 2 by SPPS

Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Synthon1-H)-Leu-OH

Equipment:

Peptide-synthesizer equipped with a 250 ml glass-reactor with frit andmanifold for automatic solvent delivery, shaking and sucking offreagents.

2A(1) Synthesis of Fmoc-Thr-Leu-Trt-Tentagel-S

Fmoc-Leu-Trt-Tentagel-S-Resin (18.7 g loading 0.37 mmol/g (supplied byRapp Polymere GmBH, Tübingen/Germany)) were swollen in DMF by shakingfor 30 min.

The Fmoc protecting group was cleaved by two subsequent treatments with20% piperidine in DMF for 5 min and 15 min respectively. After resinwashing by several alternating washes with DMF and isopropanol thecomplete removal of bases was checked with the absence of a pink colorafter Phenolphthalein and water addition to the last washing step.

4.7 g of Fmoc-Thr-OH, 5.26 g of HATU and 1.8 g of DIPEA were dissolvedin 50 ml of DMF. After 5 min of stirring additional 1.8 g of DI PEA wereadded. After checking the pH (>11) the mixture was added to thedeprotected resin and shaken for 2 h. The performed Kaiser test was OKand the resin was washed by several alternating washes with DMF andisopropanol. After drying the resin weight was 19.01 g. A small samplewas cleaved and checked by HPLC. A single main peak showed a successfulconversion.

2A(2) Synthesis of Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S

19.01 g Fmoc-Thr-Leu-Trt-Tentagel-S-Resin (6.6 mmol) were pre-swollen inDMF and the Fmoc protecting group was cleaved by two subsequenttreatments of 20% piperidine in DMF for 5 min and 15 min respectively.After resin washing by several alternating washes with DMF andisopropanol the complete removal of bases was checked with the absenceof a pink color after Phenolphthalein and water addition to the lastwashing step.

8.07 g of Fmoc-Gln(Trt)-OH, 5.01 g of HATU and 3.4 g of DIPEA weredissolved in 50 ml of DMF. After checking the pH (>11) the mixture wasadded to the deprotected resin and shaken for 1.5 h. The performedKaiser test was OK and the resin was washed by several alternatingwashes with DMF and isopropanol. The resulting resin was directly usedin the following step below, only a small sample was cleaved and checkedby HPLC. A single main peak showed successful conversion.

2A(3) Synthesis of Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S

The Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S resin from above was re-swollenin DMF by shaking in DMF for 10 min. The Fmoc protecting group wascleaved by two subsequent treatments with 20% piperidine in DMF for 5min and 15 min respectively. The resin was washed by several alternatingwashes with DMF and isopropanol. Phenolphthalein and water were added toa sample of the final wash solution. The absence of pink color provedsuccessful removal of piperidine.

0.774 g of acetic-acid, 6.707 g of PyBOP and 3.33 g of DIPEA weredissolved in 50 ml of DMF. After checking the pH (>11) the mixture wasadded to the deprotected resin and shaken for 2.5 h. The performedKaiser test was OK and the resin was washed by several alternatingwashes with DMF and isopropanol. The resulting resin was directly usedin the following step below, only a small sample was cleaved and checkedbe HPLC. A single main peak showed successful conversion.

2A(4) Synthesis of Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S(Previously Named:Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S)(Side-Chain Esterification)

The Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S resin from above was re-swollenin DMF by shaking in DMF for 10 min.

8.7 g of Fmoc-Val-OH and 8.3 g of DIPEA were dissolved in 25 ml of DCM.In parallel 2.76 g of MSNT were dissolved in another 25 ml of DCM. Bothsolutions were combined and after 3 min pre-activation put to thepeptide resin and shaken for 2 h. The resin was washed by severalalternating washes with DMF and isopropanol. The resulting resin wasdirectly used in the following step below, only a small sample wascleaved and checked be HPLC. A single main peak showed successfulconversion.

2A(5) Synthesis ofAc-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Fmoc)-Leu-Trt-Tentagel-S (PreviouslyNamed: Ac-Gln(Trt)-Thr(Val-N-me-Tyr(tBu)-Fmoc)-Leu-Trt-Tentagel-S)

The Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S resin from above wasre-swollen in DMF by shaking in DMF for 10 min.

The Fmoc protecting group was cleaved by two subsequent treatments with20% piperidine in DMF for 5 min and 15 min respectively. The resin waswashed by several alternating washes with DMF and isopropanol.Phenolphthalein and water were added to a sample of the final washsolution. The absence of pink color proved successful removal ofpiperidine.

6.1 g of Fmoc-N-Me-Tyr(tBu)-OH, 4.8 g of HATU and 3.3 g of DIPEA weredissolved in 50 ml of DMF. After checking the pH (>11) the mixture wasadded to the deprotected resin and shaken for 2.5 h. The performedKaiser test was OK and the resin was washed by several alternatingwashes with DMF and isopropanol. The resulting resin was directly usedin the following step below, only a small sample was cleaved and checkedbe HPLC. A single main peak showed successful conversion.

2A(6) Synthesis ofAc-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Fmoc)-Leu-Trt-Tentagel-S (PreviouslyNamed: Ac-Gln(Trt)-Thr(Val-N-me-Tyr(tBu)-Phe-Fmoc)-Leu-Trt-Tentagel-S)

Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Fmoc)-Leu-Trt-Tentagel-S resin from abovewas re-swollen in DMF by shaking in DMF for 10 min.

The Fmoc protecting group was cleaved by two subsequent treatments with20% piperidine in DMF for 5 min and 15 min respectively. The resin waswashed by several alternating washes with DMF and isopropanol.Phenolphthalein and water were added to a sample of the final washsolution. The absence of pink color proved successful removal ofpiperidine.

9.66 g of Fmoc-Phe-OH, 9.48 g of HATU and 6.4 g of DIPEA were dissolvedin 100 ml of DMF. After checking the pH (>11) the mixture was added tothe deprotected resin and shaken for 2 h. The performed Kaiser test wasOK and the resin was washed by several alternating washes with DMF andisopropanol. The resulting resin was directly used in the following stepbelow, only a small sample was cleaved and checked be HPLC. A singlemain peak showed successful conversion.

2A(7) Synthesis of Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Synthon1-H)-Leu-OH(Previously named:Ac-Gln(Trt)-Thr(Val-N-me-Tyr(tBu)-Phe-Synthon1-H)-Leu-OH) (=Precursorpeptide 2)

Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Fmoc)-Leu-Trt-Tentagel-S resin fromabove was re-swollen in DMF by shaking in DMF for 10 min.

The Fmoc protecting group was cleaved by two subsequent treatments with20% piperidine in DMF for 5 min and 15 min respectively. The resin waswashed by several alternating washes with DMF and isopropanol.Phenolphthalein and water were added to a sample of the final washsolution. The absence of pink color proved successful removal ofpiperidine.

6.77 g of Synthon1, 5.9 g of PyBOP and 2.95 g of DIPEA were dissolved in100 ml of DMF. After checking the pH (>11) the mixture was added to thedeprotected resin and shaken for 2 h. The performed Kaiser test was OKand the resin washed by several alternating washes with DMF andisopropanol. After taking a small sample for a HPLC check, the Fmocprotecting group was cleaved by two subsequent treatments with 20%piperidine in DMF for 5 min and 15 min respectively. The resin waswashed by several alternating washes with DMF and isopropanol. Finallythe resin was washed two times with DCM. Cleavage of the synthesizedpeptide from the resin was achieved by shaking in a mixture of 80% ofacetic acid in DCM over night. The resulting peptide solution wasfiltered off and the resin was washed two times with DCM. The combinedfiltrates were evaporated and finally purified by RP-chromatographyusing a gradient system. The collected fractions were analyzed by HPLCand the pure fractions were pooled, evaporated and finally lyophilized.

Yield: 3.54 g (42%) Precursor peptide 2.

HR-MS: Calculated for C₈₅H₁₀₈N₈O₁₃Si: [M+H]⁺=1477.78779. Found:[M+H]⁺=1477.78691.

2B Synthesis of Compound B

(S)-2-acetamido-N¹-((1S,2S,5S,8S,11R,12S,15S,18S,21R)-2-benzyl-21-hydroxy-5-(4-hydroxybenzyl)-15-isobutyl-8-isopropyl-4,11-dimethyl-3,6,9,13,16,22-hexaoxo-10-oxa-1,4,7,14,17-pentaazabicyclo[16.3.1]docosan-12-yl)pentanediamide

2B(0) Synthesis of Compound 9

(S)-2-acetamido-N¹-((3S,6S,9S,12S,15S,18S,19R)-9-benzyl-6-(4-(tert-butoxy)benzyl)-12-(3-((tert-butyldiphenylsilyl)oxy)propyl)-15-isobutyl-3-isopropyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-N⁵-tritylpentanediamide

The lactamization of Precursor peptide 2 to obtain Compound B wasperformed under similar conditions described for the preparation ofCompound 6. The product, Compound 9, was fully characterized by IR, NMRand MS. The spectra confirmed the proposed structure.

HR-MS: Calculated for C₈₅H₁₀₆N₈O₁₂Si: [M+H]⁺: 1459.77723; [M+NH₄]⁺:1476.80377. Found: [M+H]⁺: 1459.77719; [M+NH₄]⁺: 1476.80291.

2B(1) Synthesis of Compound 10

(S)-2-acetamido-N¹-((3S,6S,9S,12S,15S,18S,19R)-9-benzyl-6-(4-(tert-butoxy)benzyl)-12-(3-hydroxypropyl)-15-isobutyl-3-isopropyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-N⁵-tritylpentanediamide

The de-silylation reaction to obtain compound 10 was performed undersimilar conditions described for the preparation of compound 7. Theproduct was fully characterized by IR, NMR and MS. The spectra confirmedthe proposed structure.

HR-MS: Calculated for C₆₉H₈₈N₈O₁₂: [M+H]⁺: 1221.65945; [M+NH₄]⁺:1238.6860; [M+Na]⁺: 1243.64139. Found: [M+H]⁺: 1221.65894; [M+NH₄]⁺:1238.68518; {M+Na]⁺: 1243.64001.

2B(2) Synthesis of Compound 11 (Aldehyde)

(S)-2-acetamido-N¹-((3S,6S,9S,12S,15S,18S,19R)-9-benzyl-6-(4-(tert-butoxy)benzyl)-15-isobutyl-3-isopropyl-7,19-dimethyl-2,5,8,11,14,17-hexaoxo-12-(3-oxopropyl)-1-oxa-4,7,10,13,16-pentaazacyclononadecan-18-yl)-N⁵-tritylpentanediamide

The oxidation of Compound 10 to obtain Compound 11 was performed undersimilar conditions described for the preparation of Compound 8. LC-MSanalysis showed several peaks with the desired mass, indicating thepresence of a mixture of the aldehyde, 5-ring-hemiaminale and6-ring-hemiaminale. The mixture was used as such for the next step.HR-MS (major peak): Calculated for C₆₉H₈₆N₈O₁₂: [M+H]⁺: 1219.64380;[M+NH₄]⁺: 1236.67035; [M+Na]⁺: 1241.62574. Found: [M+H]⁺: 1219.64404;[M+NH₄]⁺: 1236.67053; {M+Na]⁺: 1241.62524.

2B(3) Synthesis of Compound B (Nostopeptin BN920) from Compound 11

(S)-2-acetamido-N¹-((1S,2S,5S,8S,11R,12S,15S,18S,21R)-2-benzyl-21-hydroxy-5-(4-hydroxybenzyl)-15-isobutyl-8-isopropyl-4,11-dimethyl-3,6,9,13,16,22-hexaoxo-10-oxa-1,4,7,14,17-pentaazabicyclo[16.3.1]docosan-12-yl)pentanediamide

Compound(s) 11 (1.0 g, 0.82 mmol) was dissolved in dichloromethane (200mL). Trifluoroacetic acid (57.8 g) was added at 15 to 25° C. during 10minutes and the reaction mixture was stirred for 45 hours at roomtemperature. The reaction mixture was then diluted with dichloromethane(200 mL) and water (10 mL) was added. Stirring was continued foradditional 24 hours at room temperature. The reaction mixture was pouredonto a saturated aq. NaHCO₃-solution (700 mL) within 20 minutes anddichloromethane (500 mL), isopropanol (50 mL) and water (500 mL) wereadded sequentially. The layers were separated and the aqueous layer wasextracted with a solution of isopropanol (50 mL) in dichloromethane (500mL). The layers were again separated and the aqueous layer was extractedseveral times with dichloromethane (5×300 mL). The organic layers werecombined and the solvent was evaporated under reduced pressure to obtaincrude product mixture (1.0 g), comprising the desired Nostopeptin BN920along with trityl alcohol and other byproducts of the reaction. Thecrude product was purified by silica gel chromatography withdichloromethane/isopropanol as mobile phase to obtain pure NostopeptinBN920 (212 mg, 97% (A) purity). Collection of less pure fractions gaveadditional 402 mg Product with 90% (A) purity. Yield from all fractions:614 mg (81%). The product was fully characterized by IR, NMR and MS. Thespectra confirmed the proposed structure.

HR-MS: Calculated for C₄₆H₆₄N₈O₁₂: [M+H]⁺: 921.47165; [M+NH₄]⁺:938.49820; [M+Na]⁺: 943.45359. Found: [M+H]⁺: 921.47167; [M+NH₄]⁺:938.49861; [M+Na]⁺: 943.45337.

Example 3 Shift of Equilibrium

Reaction Scheme 4 shows the presence of the dehydrate form of CompoundA. It has now been discovered that this can be converted easily (back)into Compound A using a simple procedure for hydration of the dehydrateform depicted in the following reaction scheme:

This allows to improve the yield of Compound A in any type of synthesis(be it chemical as in the present disclosure or by use of fermentationas in WO2009/024527).

For example, during cleavage of acid sensitive protecting groups in acompound comprising the ahp-subunit, e.g. Compound A in Example 1, theformation of large amounts of the corresponding dehydrated byproduct isobserved. This byproduct is usually separated e.g. by chromatography anddisposed. This leads to loss of valuable product and to low yield forthis step. For example, if the oxidation product(s) of Compound 8 aresubjected to acidic conditions to cleave the trityl- and t-butylprotecting groups (scheme 4), significant amounts ofCompound-A-dehydrate are formed as byproduct. Depending on the acidconcentration and reaction conditions, Compound-A-dehydrate might beformed even as major product in this product mixture.

For example, a ratio Compound A/Compound A-dehydrate (1:2) was observedwhen trifluoroacetic acid/dichloromethane (5:95 v/v) was used to cleavethe protecting groups after the oxidation step (Example 1).

It was therefore searched for ways to convert the dehydrate byproductinto the desired product. It has now been found that this can beachieved by acid catalyzed equilibration of the product mixture in thepresence of water under well defined conditions. Addition of water tothe reaction mixture of example 1 and subsequent stirring at roomtemperature for 19 h gave a product mixture with a ratio CompoundA/Compound A-dehydrate of ca. 96:4. Thus, addition of water to thereaction mixture after the acid-catalyzed deprotection step (scheme 4)changed the ratio of Compound A/Compound A-dehydrate from (1:2) underwater free conditions to (96:4) after water addition and equilibration.

The formation of Compound A-dehydrate from Compound A under acidicdeprotection conditions was confirmed by conversion of pure Compound Ainto Compound A-dehydrate using trifluoroacetic acid in DCM. Treatmentof Compound A with 33% (v/v) TFA in DCM for 2 h at room temperature gavea product mixture of Compound A-dehydrate/Compound A in a ratio of 78:22according to HPLC. The dehydration could be driven to >95% conversion,when water absorbing agents, such as molecular sieves were added to thereaction mixture. Thus, stirring of pure Compound A in a 1:2 mixture ofTFA/DCM in the presence of molecular sieves gave the dehydrated-productin quantitative crude yield and ca. 96 area % HPLC purity (example 3B).There were still ca. 4 area % of Compound A present in the crudeproduct.

Conversion of Compound A-dehydrate from Example 3B into Compound A wasdemonstrated by stirring Compound A-dehydrate in dichloromethane in thepresence of trifluoroacetic acid and water (Example 3C). The productthus obtained comprised 95.6 area % Compound A and only 4.4 area %Compound A-dehydrate according to HPLC.

Experimental Details for Example 3

3A

23 mg product mixture from oxidation (compound 8 and cyclic aminalsderived from it) was dissolved in DCM (5.7 mL) and TFA (0.3 mL) wasadded to the solution under intensive stirring. The reaction mixture wasstirred for 5 h at room temperature, until an IPC(HPLC) indicatedcomplete cleavage of the protecting groups. Compound A and CompoundA-dehydrate were present in the reaction mixture in a ratio of 1:2,along with additional byproducts. The reaction mixture was diluted withDCM (5.7 mL) and the intensively stirred solution was treated with water(0.23 mL). HPLC after 19 h stirring time at room temperature indicated aratio of 96:4 for Compound A/Compound A-dehydrate.

For workup, the reaction mixture was poured onto a solution of sodiumacetate (1.62 g) in water (23 mL) and ethyl acetate (35 mL) was added.The phases were separated and the organic layer was extracted with water(2×25 mL). The water layers were extracted with ethyl acetate (35 mL)and the organic layers were combined. The solvent was evaporated atreduced pressure to obtain 22 mg of crude product as a foam. The crudeproduct was purified by flash chromatography on silica gel with ethylacetate/methanol 95:5 to 90:10 to obtain Compound A in 96.8 area %purity, accompanied by 1.8 area % of the 5-ring isomer.

3B

Compound A (250 mg) was dissolved in dichloromethane (10 mL) andtrifluoroacetic acid (5 mL) was added, followed by the addition ofmolecular sieves (1 g). IPC(HPLC) after 1 h indicated a ratio of 17:83for Compound A/Compound A-dehydrate. The reaction mixture was stirredfor a total of 72 h at room temperature. For workup, the molecularsieves were removed by filtration and the solution was poured ontosaturated aq. NaHCO₃-solution. The aqueous layer was extracted withethyl acetate (50 mL). The organic phase was washed with water (20 mL)and the solvent was evaporated at reduced pressure and the residue wasdried in vacuo to obtain quantitative yield (245 mg) crude product as afoam. HPLC analysis of the crude product indicated the presence ofCompound A/Compound A-dehydrate at a ratio of ca. 4:96 area %.

HR-MS: calculated for C₄₆H₇₀N₈O₁₁ [M+H]⁺=911.52368, [M+NH4]⁺=928.55023,[M+Na]+=933.50563. Found [M+H]⁺=911.52372, [M+NH4]⁺=928.55029,[M+Na]⁺=933.50538. The structure of Compound A-dehydrate was confirmedby ¹H-NMR.

¹H-NMR (600 MHz, d₆-DMSO) δ_(H): 0.06 (3H, d, J=6.6 Hz), 0.67 (3H, t,J=7.3 Hz), 0.70 (3H, d, J=7.0 Hz), 0.77 (3H, d, J=6.6 Hz), 0.81 (1H, m),0.87 (6H, m), 1.00 (3H, d, J=7.0 Hz), 1.02 (3H, d, J=7.0 Hz), 1.06 (1H,m), 1.16 (3H, d, J=6.5 Hz), 1.17 (1H, m), 1.30 (1H, m), 1.41 (1H, m),1.53 (1H, m), 1.74 (2H, m), 1.91 (2H, m), 2.01 (1H, m), 2.11 (2H, m),2.45 (3H, m), 2.73 (3H, s), 2.74 (1H, m), 3.18 (1H, m), 4.32 (2H, m),4.50 (1H, m), 4.54 (1H, m), 4.64 (1H, d, J=9.5 Hz), 4.77 (1H, d, J=11.0Hz), 5.18 (1H, m), 5.26 (1H, m), 5.42 (1H, q, J=6.6 Hz), 6.25 (1H, d,J=7.3 Hz), 6.32 (1H, d, J=7.7 Hz), 6.67 (2H, d, J=8.4 Hz), 6.75 (1H, s),7.04 (2H, d, J=8.4 Hz), 7.25 (1H, s), 7.30 (1H, d, J=8.8 Hz), 7.89 (1H,d, J=9.2 Hz), 7.98 (1H, d, J=8.1 Hz), 8.50 (1H, d, J=8.4 Hz), 9.24 (1H,s).

3C

Compound A-dehydrate (110 mg) from example 2 was dissolved in DCM (20mL). TFA (1 g) and water (0.2 mL) were added and the reaction mixturewas stirred for 20 h at room temperature. For workup, the reactionmixture was poured onto ethyl acetate (50 mL) and the ethyl acetatesolution was extracted with a saturated aq. NaHCO₃-solution (50 mL). Theorganic layer was extracted with water (20 mL) and the solvent wasevaporated. The residue was dissolved in ethyl acetate/isopropanol 9:1(20 mL) and the solution was clear filtered over a 0.45 micrometerfilter. The solvent was evaporated under reduced pressure and theresidue was dried in vacuo at 45° C. to obtain 100 mg crude product.HPLC analysis indicated the presence of ca. 95.6 area % Compound A andca. 4.4 area % Compound A-dehydrate.

What is claimed is:
 1. A compound selected from a compound of any one ofFormulae II, III, VI, VIII and XXV:

wherein Prot is a protecting group, and Prot* is a protecting group thatcan be cleaved off selectively without affecting the other protectinggroups present and which is a C₃-C₈alk-2-enyloxycarbonyl moiety; Y ishydrogen or C₁₋₈-alkyl and X*, A₁*, R₂*, R₃*, R₅*, R₆*, and R₇ ^(*)correspond to X, A₁, R₂, R₃, R₅, R₆, and R₇ respectively, wherein A_(l)is a bivalent moiety of an amino acid with a terminal carboxy orcarbamoyl group, and is bound at its right hand side in formula I via acarbonyl to the rest of the molecule; or is C₁₋₈-alkanoyl orphosphorylated hydroxy-C₁₋₈-alkanoyl; X is bound via an N of A₁ and isacyl, or is absent if A₁ is C₁₋₈-alkanoyl or phosphorylatedhydroxy-C₁₋₈-alkanoyl; R₂ is C₁₋₈-alkyl; R₃ is the side chain ofleucine, isoleucine or valine; R₅ is the side chain of phenylalanine,leucine, isoleucine or valine; R₆ is the side chain of a hydroxy aminoacid; and R₇ is the side chain of the amino acid leucine, isoleucine orvaline; however with the proviso that reactive functional groups onthese moieties are present in unprotected or protected form, or a saltthereof.
 2. The compound according to claim 1 of Formula II:

or a salt thereof.
 3. The compound according to claim 1, having FormulaII,

wherein Prot is a protecting group, Y is methyl and X*, A₁*, R₂*, R₃*,R₅*, R₆*, and R₇* correspond to X, A₁, R₂, R₃, R₅, R₆, and R₇respectively, wherein A_(l) is a bivalent radical of L-glutamine boundvia the carbonyl of its α-carboxyl group to the amino group at the rightof A₁ in formula I and via its α-amino group to X, or is2S-(2-hydroxy-3-phosphonooxy)-propionyl; R₂ is methyl; R₃ is isopropylor isobutyl; R₅ is sec-butyl or benzyl; R₆ is 4-hydroxybenzyl; R₇ isisopropyl or sec-butyl, and X is acetyl or isobutyryl, or is absent ifA_(l) is 2S-(2-hydroxy-3-phosphonooxy)-propionyl, however with theproviso that reactive functional groups on these moieties are present inunprotected or protected form, or a salt thereof.
 4. The compoundaccording to claim 1 having Formula III,

or a salt thereof.
 5. The compound according to claim 1 having FormulaIII,

wherein Y is methyl and X*, A₁*, R₂*, R₃*, R₅*, R₆*, and R₇* correspondto X, A₁, R₂, R₃, R₅, R₆, and R₇ respectively, wherein A₁ is a bivalentradical of L-glutamine bound via the carbonyl of its α-carboxyl group tothe amino group at the right of A₁ in formula I and via its α-aminogroup to X, or is 2S-(2-hydroxy-3-phosphonooxy)-propionyl; R₂ is methyl;R₃ is isopropyl or isobutyl; R₅ is sec-butyl or benzyl; R₆ is4-hydroxybenzyl; R₇ is isopropyl or sec-butyl; and X is acetyl orisobutyryl, or is absent if A_(l) is2S-(2-hydroxy-3-phosphonooxy)-propionyl, however with the proviso thatreactive functional groups on these moieties are present in unprotectedor protected form, or a salt thereof.
 6. The compound according to claim1 having Formula VI

or a salt thereof.
 7. The compound according to claim 1 having FormulaVI

wherein Prot is a protecting group, Y is methyl and R₂*, R₃*, R₅*, R₆*,and R₇* correspond to R₂, R₃, R₅, R₆, and R₇ respectively, wherein R₂ ismethyl; R₃ is isopropyl or isobutyl; R₅ is sec-butyl or benzyl; R₆ is4-hydroxybenzyl; and R₇ is isopropyl or sec-butyl, however with theproviso that reactive functional groups on these moieties are present inunprotected or protected form, or a salt thereof.
 8. The compound ofclaim 1 having Formula VIII

or a salt thereof.
 9. The compound according to claim 1 having FormulaVIII

wherein Prot is a protecting group, Prot* is a protecting group that canbe cleaved off selectively without affecting the other protecting groupspresent, Y is methyl and R₂*, R₃*, R₅*, R₆*, and R₇* correspond to R₂,R₃, R₅, R₆, and R₇respectively, wherein R₂ is methyl; R₃ is isopropyl orisobutyl; R₅ is sec-butyl or benzyl; R₆ is 4-hydroxybenzyl; and R₇ isisopropyl or sec-butyl, however with the proviso that reactivefunctional groups on these moieties are present in unprotected orprotected form, or a salt thereof.
 10. The compound according to claim 1having Formula XXV

or a salt thereof.
 11. The compound according to claim 1 having FormulaXXV

wherein Prot is a protecting group, Y is methyl and X*, A₁*, R₂*, R₃*,R₅*, R₆*, and R₇* correspond to X, A₁, R₂, R₃, R₅, R₆, and R₇respectively, wherein A_(l) is a bivalent radical of L-glutamine boundvia the carbonyl of its α-carboxyl group to the amino group at the rightof A_(l) in formula I and via its α-amino group to X, or is2S-(2-hydroxy-3-phosphonooxy)-propionyl; R₂ is methyl; R₃ is isopropylor isobutyl; R₅ is sec-butyl or benzyl; R₆ is 4-hydroxybenzyl; R₇ isisopropyl or sec-butyl, and X is acetyl or isobutyryl, or is absent ifA_(l) is 2S-(2-hydroxy-3-phosphonooxy)-propionyl, however with theproviso that reactive functional groups on these moieties are present inunprotected or protected form, or a salt thereof.
 12. The compoundaccording to claim 1 selected from

or a salt thereof.